Claims:

1-42. (canceled)

43. A method of screening compounds to identify a candidate compound for
treating overactive bladder, the method comprising:a) contacting a BACH
GPCR polypeptide with a compound andb) determining whether the compound
binds to and inhibits activation of the BACH GPCR polypeptide,wherein if
the compound binds to and inhibits activation of the BACH GPCR
polypeptide, identifying the compound as a candidate compound for
treating overactive bladder, wherein the overactive bladder is associated
with the activation of the BACH GPCR polypeptide.

45. The method according to claim 43, wherein the compound is exposed to a
cell expressing a BACH GPCR polypeptide.

46. The method according to claim 45, wherein a change in cellular cyclic
AMP (cAMP) or calcium levels is detected.

47. The method according to claim 46, wherein a decrease in cellular
cyclic AMP levels is detected, thereby identifying an antagonist of BACH
GPCR polypeptide.

48. The method according to claim 46, wherein an increase in cyclic AMP
levels is detected, thereby identifying an agonist of BACH GPCR
polypeptide.

49. A method of screening compounds to identify a candidate compound for
treating overactive bladder, the method comprising:a) contacting a BACH
GPCR polypeptide with a compound and determining whether the compound
binds to and inhibits activation of the BACH GPCR polypeptide;b)
administering the compound that binds to and inhibits activation of the
BACH GPCR polypeptide to an animal; andc) determining whether the animal
exhibits decreased micturition, thereby identifying a candidate compound
for treating overactive bladder, wherein the overactive bladder is
associated with the activation of the BACH GPCR polypeptide.

50. The method according to claim 49, wherein the decreased micturition is
selected from an increase in micturition interval, and an increase in
micturition volume.

52. The method according to claim 49, wherein the animal is a wild type
animal.

53. The method according to claim 49, wherein the animal is a rodent.

54. The method according to claim 49, wherein the animal is a mouse.

Description:

FIELD

[0001]This invention relates to newly identified nucleic acids,
polypeptides encoded by them and to their production and use. More
particularly, the nucleic acids and polypeptides of the present invention
relate to a G-protein coupled receptor (GPCR), hereinafter referred to as
"BACH GPCR", and members of the purinoceptor family of GPCRs. The
invention also relates to inhibiting or activating the action of such
nucleic acids and polypeptides.

[0003]For example, in one form of signal transduction, the effect of
hormone binding is activation of the enzyme adenylate cyclase inside the
cell. Enzyme activation by hormones is dependent on the presence of the
nucleotide, GTP. GTP also influences hormone binding. A G-protein
connects the hormone receptor to adenylate cyclase. G-protein is shown to
exchange GTP for bound GDP when activated by a hormone receptor. The GTP
carrying form then binds to activated adenylate cyclase. Hydrolysis of
GTP to GDP, catalysed by the G-protein itself, returns the G-protein to
its basal, inactive form. Thus, the G-protein serves a dual role, as an
intermediate that relays the signal from receptor to effector, and as a
clock that controls the duration of the signal.

[0004]The membrane protein gene superfamily of G-protein coupled receptors
(GPCRs) has been characterised as having seven putative transmembrane
domains. The domains are believed to represent transmembrane
α-helices connected by extracellular or cytoplasmic loops.
G-protein coupled receptors include a wide range of biologically active
receptors, such as hormone, viral, growth factor and neuroreceptors.

[0005]G-protein coupled receptors (also known as 7TM receptors) have been
characterised as including these seven conserved hydrophobic stretches of
about 20 to 30 amino acids, connecting at least eight divergent
hydrophilic loops. The G-protein family of coupled receptors includes
dopamine receptors which bind to neuroleptic drugs used for treating
psychotic and neurological disorders. Other examples of members of this
family include, but are not limited to, calcitonin, adrenergic,
endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin,
histamine, thrombin, kinin, follicle stimulating hormone, opsins,
endothelial differentiation gene-1, rhodopsins, odorant, and
cytomegalovirus receptors.

[0006]Most G-protein coupled receptors have single conserved cysteine
residues in each of the first two extracellular loops which form
disulphide bonds that are believed to stabilise functional protein
structure. The 7 transmembrane regions are designated as TM1, TM2, TM3,
TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.

[0007]Phosphorylation and lipidation (pamitylation or farnesylation) of
cysteine residues can influence signal transduction of some G-protein
coupled receptors. Most G-protein coupled receptors contain potential
phosphorylation sites within the third cytoplasmic loop and/or the
carboxy terminus For several G-protein coupled receptors, such as the
β-adrenoreceptor, phosphorylation by protein kinase A and/or
specific receptor kinases mediates receptor desensitization. For some
receptors, the ligand binding sites of G-protein coupled receptors are
believed to comprise hydrophilic sockets formed by several G-protein
coupled receptor transmembrane domains, the sockets being surrounded by
hydrophobic residues of the G-protein coupled receptors. The hydrophilic
side of each G-protein coupled receptor transmembrane helix is thought to
face inward and form a polar ligand binding site. TM3 has been implicated
in several G-protein coupled receptors as having a ligand binding site,
such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6
or TM7 phenylalanines or tyrosines are also implicated in ligand binding.

[0008]G-protein coupled receptors can be intracellularly coupled by
heterotrimeric G-proteins to various intracellular enzymes, ion channels
and transporters (see, Johnson et al., Endoc. Rev., 1989, 10: 317-331).
Different G-protein α-subunits preferentially stimulate particular
effectors to modulate various biological functions in a cell.
Phosphorylation of cytoplasmic residues of G-protein coupled receptors
has been identified as an important mechanism for the regulation of
G-protein coupling of some G-protein coupled receptors. G-protein coupled
receptors are found in numerous sites within a mammalian host. Over the
past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7
TM) receptors have been successfully introduced onto the market.

[0010]According to a first aspect of the present invention, we provide a
BACH GPCR polypeptide comprising the amino acid sequence shown in SEQ ID
NO. 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.

[0011]There is provided, according to a second aspect of the present
invention, a nucleic acid capable of encoding a polypeptide according to
the first aspect of the invention. Preferably, the nucleic acid comprises
the nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 SEQ ID NO:
4, SEQ ID NO: 10 or a homologue, variant or derivative thereof.

[0012]We provide, according to a third aspect of the present invention, a
polypeptide comprising a fragment of a polypeptide according to the first
aspect of the invention.

[0013]Preferably, such a fragment containing polypeptide comprises one or
more regions which are homologous between SEQ ID No. 3 and SEQ ID No. 5,
or which comprises one or more regions which are heterologous between SEQ
ID No. 3 and SEQ ID No. 5.

[0014]As a fourth aspect of the present invention, there is provided a
nucleic acid capable of encoding a polypeptide according to the third
aspect of the invention.

[0015]We provide, according to a fifth aspect of the present invention, a
vector comprising a nucleic acid according to the second or fourth aspect
of the invention.

[0016]The present invention, in a sixth aspect, provides a host cell
comprising a nucleic acid according to the second or fourth aspect of the
invention, or vector according to the fifth aspect of the invention.

[0017]In a seventh aspect of the present invention, there is provided a
transgenic non-human animal comprising a nucleic acid according to the
second or fourth aspect of the invention or a vector according to the
fifth aspect of the invention. Preferably, the transgenic non-human
animal is a mouse.

[0018]According to an eighth aspect of the present invention, we provide
use of a polypeptide according to the first or third aspect of the
invention in a method of identifying compound which is capable of
interacting specifically with a G protein coupled receptor.

[0019]We provide, according to a ninth aspect of the invention, use of a
transgenic non-human animal according to the seventh aspect of the
invention in a method of identifying a compound which is capable of
interacting specifically with a G protein coupled receptor.

[0020]There is provided, in accordance with a tenth aspect of the present
invention, a method for identifying an antagonist of a BACH GPCR, the
method comprising contacting a cell which expresses BACH receptor with a
candidate compound and determining whether the level of cyclic AMP (cAMP)
in said cell is lowered as a result of said contacting.

[0021]As an eleventh aspect of the invention, we provide a method for
identifying a compound capable of lowering the endogenous level of cyclic
AMP in a cell which method comprises contacting a cell which expresses a
BACH GPCR with a candidate compound and determining whether the level of
cyclic AMP (cAMP) in said cell is lowered as a result of said contacting.

[0022]According to a twelfth aspect of the invention, we provide a method
for identifying a compound capable of binding to a BACH GPCR polypeptide,
the method comprising contacting a BACH GPCR polypeptide with a candidate
compound and determining whether the candidate compound binds to the BACH
GPCR polypeptide.

[0023]We provide, according to a thirteenth aspect of the invention, there
is provided a compound identified by a method according to any of the
eighth to twelfth aspects of the invention.

[0024]According to a fourteenth aspect of the present invention, we
provide a compound capable of binding specifically to a polypeptide
according to the first or third aspect of the invention.

[0025]There is provided, according to a fifteenth aspect of the present
invention, use of a polypeptide according to the first or third aspect of
the invention, or part thereof; or a nucleic acid according to the second
or fourth aspect of the invention, or part thereof, in a method for
producing antibodies.

[0026]We provide, according to a sixteenth aspect of the present
invention, an antibody capable of binding specifically to a polypeptide
according to the first or third aspect of the invention, or part thereof;
or a polypeptide encoded by a nucleic acid according to the second or
fourth aspect of the invention, or part thereof;.

[0027]As a seventeenth aspect of the present invention, there is provided
a pharmaceutical composition comprising any one or more of the following:
a polypeptide according to the first or third aspect of the invention, or
part thereof; a polypeptide encoded by a nucleic acid according to the
second or fourth aspect of the invention, or part thereof; a vector
according to the fifth aspect of the invention; a cell according to the
sixth aspect of the invention; a compound according to the thirteenth or
fourteenth aspect of the invention; and an antibody according to the
sixteenth aspect of the invention, together with a pharmaceutically
acceptable carrier or diluent.

[0028]We provide, according to a eighteenth aspect of the present
invention, a vaccine composition comprising any one or more of the
following: a polypeptide according to the first or third aspect of the
invention, or part thereof; a polypeptide encoded by a nucleic acid
according to the second or fourth aspect of the invention, or part
thereof; a vector according to the fifth aspect of the invention; a cell
according to the sixth aspect of the invention; a compound according to
the thirteenth or fourteenth aspect of the invention; and an antibody
according to the sixteenth aspect of the invention.

[0029]According to an nineteenth aspect of the present invention, we
provide a diagnostic kit for a disease or susceptibility to a disease
comprising any one or more of the following: a polypeptide according to
the first or third aspect of the invention, or part thereof; a
polypeptide encoded by a nucleic acid according to the second or fourth
aspect of the invention, or part thereof; a vector according to the fifth
aspect of the invention; a cell according to the sixth aspect of the
invention; a compound according to the thirteenth or fourteenth aspect of
the invention; and an antibody according to the sixteenth aspect of the
invention.

[0030]We provide, according to a twentieth aspect of the invention, a
method of treating a patient suffering from a disease associated with
enhanced activity of a BACH GPCR, which method comprises administering to
the patient an antagonist of BACH GPCR.

[0031]There is provided, in accordance with a twenty-first aspect of the
present invention, a method of treating a patient suffering from a
disease associated with reduced activity of a BACH GPCR, which method
comprises administering to the patient an agonist of BACH GPCR.

[0033]According to a twenty-second aspect of the present invention, we
provide a method for treating and/or preventing a disease in a patient,
which comprises the step of administering any one or more of the
following to the patient: a polypeptide according to the first or third
aspect of the invention, or part thereof; a polypeptide encoded by a
nucleic acid according to the second or fourth aspect of the invention,
or part thereof; a vector according to the fifth aspect of the invention;
a cell according to the sixth aspect of the invention; a compound
according to the thirteenth or fourteenth aspect of the invention; and an
antibody according to the sixteenth aspect of the invention; a
pharmaceutical composition according to the seventeenth aspect of the
invention; and a vaccine according to the eighteenth aspect of the
invention, to the subject.

[0034]There is provided, according to a twenty-third aspect of the present
invention, an agent comprising a polypeptide according to the first or
third aspect of the invention, or part thereof; a polypeptide encoded by
a nucleic acid according to the second or fourth aspect of the invention,
or part thereof; a vector according to the fifth aspect of the invention;
a cell according to the sixth aspect of the invention; a compound
according to the thirteenth or fourteenth aspect of the invention; and an
antibody according to the sixteenth aspect of the invention, said agent
for use in a method of treatment or prophylaxis of disease.

[0035]We provide, according to a twenty-fourth aspect of the present
invention, use of a polypeptide according to the first or third aspect of
the invention, or part thereof; a polypeptide encoded by a nucleic acid
according to the second or fourth aspect of the invention, or part
thereof; a vector according to the fifth aspect of the invention; a cell
according to the sixth aspect of the invention; a compound according to
the thirteenth or fourteenth aspect of the invention; and an antibody
according to the sixteenth aspect of the invention, for the preparation
of a pharmaceutical composition for the treatment or prophylaxis of a
disease.

[0036]As a twenty-fifth aspect of the present invention, there is provided
non-human transgenic animal, characterized in that the transgenic animal
comprises an altered BACH gene. Preferably, the alteration is selected
from the group consisting of: a deletion of BACH, a mutation in BACH
resulting in loss of function, introduction of an exogenous gene having a
nucleotide sequence with targeted or random mutations into BACH,
introduction of an exogenous gene from another species into BACH, and a
combination of any of these.

[0037]We provide, according to a twenty-sixth aspect of the present
invention, a non-human transgenic animal having a functionally disrupted
endogenous BACH gene, in which the transgenic animal comprises in its
genome and expresses a transgene encoding a heterologous BACH protein.

[0038]The present invention, in a twenty-seventh aspect, provides a
nucleic acid construct for functionally disrupting a BACH gene in a host
cell, the nucleic acid construct comprising: (a) a non-homologous
replacement portion; (b) a first homology region located upstream of the
non-homologous replacement portion, the first homology region having a
nucleotide sequence with substantial identity to a first BACH gene
sequence; and (c) a second homology region located downstream of the
non-homologous replacement portion, the second homology region having a
nucleotide sequence with substantial identity to a second BACH gene
sequence, the second BACH gene sequence having a location downstream of
the first BACH gene sequence in a naturally occurring endogenous BACH
gene.

[0039]According to a twenty-eighth aspect of the present invention, we
provide a process for producing a BACH GPCR polypeptide, the method
comprising culturing a host cell according to the sixth aspect of the
invention under conditions in which a nucleic acid encoding a BACH GPCR
polypeptide is expressed.

[0040]There is provided, according to a twenty-ninth aspect of the present
invention, a method of detecting the presence of a nucleic acid according
to the second or fourth aspect of the invention in a sample, the method
comprising contacting the sample with at least one nucleic acid probe
which is specific for said nucleic acid and monitoring said sample for
the presence of the nucleic acid.

[0041]We provide, according to a thirtieth aspect of the present
invention, a method of detecting the presence of a polypeptide according
to the first or third aspect of the invention in a sample, the method
comprising contacting the sample with an antibody according to the
sixteenth aspect of the invention and monitoring said sample for the
presence of the polypeptide.

[0042]As a thirty-first aspect of the present invention, there is provided
a method of diagnosis of a disease or syndrome caused by or associated
with increased, decreased or otherwise abnormal expression of BACH GPCR,
the method comprising the steps of: (a) detecting the level or pattern of
expression of BACH GPCR in an animal suffering or suspected to be
suffering from such a disease; and (b) comparing the level or pattern of
expression with that of a normal animal.

[0044]In another embodiment, the disease is selected from the group
consisting of: dementia, dyslexia, dyskinesias, tremor, Parkinson's,
benign essential tremor, chorea, epilepsy and ballismus, for example
occurring through stroke, trauma, degeneration or malignancy.

[0045]In a further embodiment, the disease is selected from the group
consisting of: dry-eye disorders, cystic fibrosis, hyperactive bladder,
hypercholesterolaemia, dislipdaemias and obesity.

[0046]These and other embodiments of the invention will be described in
further detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1 is a diagram showing the results of analysis of the human
BACH polypeptide (SEQ ID NO: 3) using the HMM structural prediction
software of pfam (http://www.sanger.ac.uk/Software/Pfam/search.shtml).

[0049]FIG. 3 shows a restriction map of vector pTK3IBLMNL used in the
construction of transgenic BACH knock-out mice.

[0050]FIG. 4 shows the structure of an allele of BACH in which the
transmembrane regions are replaced by a lacZ reporter gene and a
selectable marker; BACH knock-out mouse may be prepared comprising this
allele using the methods described in the Examples.

[0057]A genomic sequence of mouse BACH is shown in the section headed
"BACH Genomic Sequence" (SEQ ID NO: 10). Such a sequence may be used to
prepare BACH knock out mice as described below and in the Examples.

DETAILED DESCRIPTION

BACH GPCR

[0058]Our invention relates in general to a novel G-Protein Coupled
Receptor (GPCR), in particular, an orphan purinoceptor type G-protein
coupled receptor, which we refer to as BACH GPCR, as well as homologues,
variants or derivatives thereof.

[0059]BACH is structurally related to other proteins of the G-protein
coupled receptor family, as shown by the results of sequencing the
amplified cDNA products encoding human BACH. The cDNA sequence of SEQ ID
NO: 1 contains an open reading flame (SEQ ID NO: 2, nucleotide numbers 23
to 800) encoding a polypeptide of 372 amino acids shown in SEQ ID NO: 3.
Human BACH is found to map to Homo sapiens chromosome 12p13.3.

[0063]Human and mouse BACH GPCR are therefore members of a large family of
G Protein Coupled Receptors (GPCRs).

Expression Profile of BACH

[0064]Polymerase chain reaction (PCR) amplification of BACH cDNA detects
expression of BACH to varying abundance in human spleen, heart, brain and
liver. An expression profile of BACH GPCR is shown in FIG. 2. Using BACH
cDNA of SEQ ID NO: 1 to search the human EST data sources by BLASTN,
identities are found in cDNA derived from libraries originating from
B-cells from chronic lymphotic leukemia (Accession #AI492234), germinal
center B cell (Accession #AA769338) and testis (Accession #AL042117).
This indicates that BACH is expressed in these normal or abnormal
tissues. Accordingly, the BACH polypeptides, nucleic acids, probes,
antibodies, expression vectors and ligands are useful for detection,
diagnosis, treatment and other assays for diseases associated with over-,
under- and abnormal expression of BACH GPCR in these and other tissues.

[0065]As shown in the Examples, expression data for BACH may also be
obtained by use of a knock out mouse for BACH, in which a lacZ reporter
gene is integrated into the endogenous BACH gene, and staining for
expression of β-galactocidase. Sites of endogenous BACH promoter
activity drive expression of the reporter and are visualised, for example
histochemically, resulting in blue cells in sites of expression. This
pattern faithfully represents the expression pattern of the endogenous
BACH transcripts. Staining patterns of lacZ showing the expression of
BACH are shown in the Figures.

[0066]The above lacZ reporter staining, Northern blot analysis and RT-PCR
experiments show that BACH is expressed in the following tissues: Brain,
more specifically surface of the cerebellum; Spinal column, more
specifically Substancia gelatinosa (caudal/sacral areas); Dorsal root
ganglia, more specifically neurones of the A-δ fibre and C fibre
class; Trigeminal ganglion and trigeminal nucleus and Cranial nerve 8;
Eye, more specifically cells of the conjuctiva; Urinary bladder; Gall
bladder; Tongue; Skin, particularly around hair follicles and in the
nasal region; pleura and surface of lungs; Salivary glands, regions of
submaxillary salivary glands; Gut, more specifically oesophagus, stomach,
villi of the small intestine, colon and rectum (crypts); and Fat and
pericardium surrounding the heart.

[0068]As used here, the term "BACH GPCR polypeptide" is intended to refer
to a polypeptide comprising the amino acid sequence shown in SEQ ID No. 3
or SEQ ID NO: 5, or a homologue, variant or derivative thereof.
Preferably, the polypeptide comprises or is a homologue, variant or
derivative of the sequence shown in SEQ ID NO: 3.

[0069]"Polypeptide" refers to any peptide or protein comprising two or
more amino acids joined to each other by peptide bonds or modified
peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both
short chains, commonly referred to as peptides, oligopeptides or
oligomers, and to longer chains, generally referred to as proteins.
Polypeptides may contain amino acids other than the 20 gene-encoded amino
acids.

[0070]"Polypeptides" include amino acid sequences modified either by
natural processes, such as post-translational processing, or by chemical
modification techniques which are well known in the art. Such
modifications are well described in basic texts and in more detailed
monographs, as well as in a voluminous research literature. Modifications
can occur anywhere in a polypeptide, including the peptide backbone, the
amino acid side-chains and the amino or carboxyl termini It will be
appreciated that the same type of modification may be present in the same
or varying degrees at several sites in a given polypeptide. Also, a given
polypeptide may contain many types of modifications.

[0072]The terms "variant", "homologue", "derivative" or "fragment" in
relation to the present invention include any substitution of, variation
of, modification of, replacement of, deletion of or addition of one (or
more) amino acid from or to a sequence. Unless the context admits
otherwise, references to "BACH" and "BACH GPCR" include references to
such variants, homologues, derivatives and fragments of BACH.

[0073]Preferably, as applied to BACH, the resultant amino acid sequence
has GPCR activity, more preferably having at least the same activity of
the BACH GPCR shown as SEQ ID NO: 3 or SEQ ID NO: 5. In particular, the
term "homologue" covers identity with respect to structure and/or
function providing the resultant amino acid sequence has GPCR activity.
With respect to sequence identity (i.e. similarity), preferably there is
at least 70%, more preferably at least 75%, more preferably at least 85%,
even more preferably at least 90% sequence identity. More preferably
there is at least 95%, more preferably at least 98%, sequence identity.
These terms also encompass polypeptides derived from amino acids which
are allelic variations of the BACH GPCR nucleic acid sequence.

[0074]Where reference is made to the "receptor activity" or "biological
activity" of a receptor such as BACH GPCR, these terms are intended to
refer to the metabolic or physiological function of the BACH receptor,
including similar activities or improved activities or these activities
with decreased undesirable side effects. Also included are antigenic and
immunogenic activities of the BACH receptor. Examples of GPCR activity,
and methods of assaying and quantifying these activities, are known in
the art, and are described in detail elsewhere in this document.

[0075]As used herein a "deletion" is defined as a change in either
nucleotide or amino acid sequence in which one or more nucleotides or
amino acid residues, respectively, are absent. As used herein an
"insertion" or "addition" is that change in a nucleotide or amino acid
sequence which has resulted in the addition of one or more nucleotides or
amino acid residues, respectively, as compared to the naturally occurring
substance. As used herein "substitution" results from the replacement of
one or more nucleotides or amino acids by different nucleotides or amino
acids, respectively.

[0076]BACH polypeptides according to the present invention may also have
deletions, insertions or substitutions of amino acid residues which
produce a silent change and result in a functionally equivalent amino
acid sequence. Deliberate amino acid substitutions may be made on the
basis of similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having similar
hydrophilicity values include leucine, isoleucine, valine, glycine,
alanine, asparagine, glutamine, serine, threonine, phenylalanine, and
tyrosine.

[0077]Conservative substitutions may be made, for example according to the
table below Amino acids in the same block in the second column and
preferably in the same line in the third column may be substituted for
each other:

[0078]BACH polypeptides of the invention may further comprise heterologous
amino acid sequences, typically at the N-terminus or C-terminus,
preferably the N-terminus Heterologous sequences may include sequences
that affect intra or extracellular protein targeting (such as leader
sequences). Heterologous sequences may also include sequences that
increase the immunogenicity of the polypeptide of the invention and/or
which facilitate identification, extraction and/or purification of the
polypeptides. Another heterologous sequence that is particularly
preferred is a polyamino acid sequence such as polyhistidine which is
preferably N-terminal. A polyhistidine sequence of at least 10 amino
acids, preferably at least 17 amino acids but fewer than 50 amino acids
is especially preferred.

[0079]The BACH GPCR polypeptides may be in the form of the "mature"
protein or may be a part of a larger protein such as a fusion protein. It
is often advantageous to include an additional amino acid sequence which
contains secretory or leader sequences, pro-sequences, sequences which
aid in purification such as multiple histidine residues, or an additional
sequence for stability during recombinant production.

[0080]BACH polypeptides of the invention are advantageously made by
recombinant means, using known techniques. However they may also be made
by synthetic means using techniques well known to skilled persons such as
solid phase synthesis. Polypeptides of the invention may also be produced
as fusion proteins, for example to aid in extraction and purification.
Examples of fusion protein partners include glutathione-S-transferase
(GST), 6× His, GAL4 (DNA binding and/or transcriptional activation
domains) and β-galactosidase. It may also be convenient to include a
proteolytic cleavage site between the fusion protein partner and the
protein sequence of interest to allow removal of fusion protein
sequences, such as a thrombin cleavage site. Preferably the fusion
protein will not hinder the function of the protein of interest sequence.

[0081]BACH polypeptides of the invention may be in a substantially
isolated form. This term is intended to refer to alteration by the hand
of man from the natural state. If an "isolated" composition or substance
occurs in nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide, nucleic acid or a
polypeptide naturally present in a living animal is not "isolated," but
the same polynucleotide, nucleic acid or polypeptide separated from the
coexisting materials of its natural state is "isolated", as the term is
employed herein.

[0082]It will however be understood that the BACH GPCR protein may be
mixed with carriers or diluents which will not interfere with the
intended purpose of the protein and still be regarded as substantially
isolated. A polypeptide of the invention may also be in a substantially
purified form, in which case it will generally comprise the protein in a
preparation in which more than 90%, for example, 95%, 98% or 99% of the
protein in the preparation is a BACH GPCR polypeptide of the invention.

[0083]The present invention also relates to peptides comprising a portion
of a BACH polypeptide according to the invention. Thus, fragments of BACH
GPCR and its homologues, variants or derivatives are included. The
peptides of the present invention may be between 2 and 200 amino acids,
preferably between 4 and 40 amino acids in length. The peptide may be
derived from a BACH GPCR polypeptide as disclosed here, for example by
digestion with a suitable enzyme, such as trypsin. Alternatively the
peptide, fragment, etc may be made by recombinant means, or synthesised
synthetically,

[0084]The term "peptide" includes the various synthetic peptide variations
known in the art, such as a retroinverso D peptides. The peptide may be
an antigenic determinant and/or a T-cell epitope. The peptide may be
immunogenic in vivo. Preferably the peptide is capable of inducing
neutralising antibodies in vivo.

[0085]By aligning BACH GPCR sequences from different species, it is
possible to determine which regions of the amino acid sequence are
conserved between different species ("homologous regions"), and which
regions vary between the different species ("heterologous regions").

[0086]The BACH polypeptides according to the invention may therefore
comprise a sequence which corresponds to at least part of a homologous
region. A homologous region shows a high degree of homology between at
least two species. For example, the homologous region may show at least
70%, preferably at least 80%, more preferably at least 90%, even more
preferably at least 95% identity at the amino acid level using the tests
described above. Peptides which comprise a sequence which corresponds to
a homologous region may be used in therapeutic strategies as explained in
further detail below. Alternatively, the BACH GPCR peptide may comprise a
sequence which corresponds to at least part of a heterologous region. A
heterologous region shows a low degree of homology between at least two
species.

BACH GPCR Polynucleotides and Nucleic Acids

[0087]This invention encompasses BACH polynucleotides, BACH nucleotides
and BACH nucleic acids, methods of production, uses of these, etc, as
described in further detail elsewhere in this document.

[0089]These terms are also intended to include a nucleic acid sequence
capable of encoding a polypeptides and/or a peptide of the present
invention, i.e., a BACH polypeptide. Thus, BACH GPCR polynucleotides and
nucleic acids comprise a nucleotide sequence capable of encoding a
polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or
SEQ ID NO: 5, or a homologue, variant or derivative thereof. Preferably,
the BACH GPCR polynucleotides and nucleic acids comprise a nucleotide
sequence capable of encoding a polypeptide comprising the amino acid
sequence shown in SEQ ID NO: 3, or a homologue, variant or derivative
thereof.

[0090]"Polynucleotide" generally refers to any polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified
RNA or DNA. "Polynucleotides" include, without limitation single- and
double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions, single- and double-stranded RNA, and RNA that is mixture of
single- and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded or a
mixture of single- and double-stranded regions. In addition,
"polynucleotide" refers to triple-stranded regions comprising RNA or DNA
or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs
containing one or more modified bases and DNAs or RNAs with backbones
modified for stability or for other reasons. "Modified" bases include,
for example, tritylated bases and unusual bases such as inosine. A
variety of modifications has been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or metabolically
modified forms of polynucleotides as typically found in nature, as well
as the chemical forms of DNA and RNA characteristic of viruses and cells.
"Polynucleotide" also embraces relatively short polynucleotides, often
referred to as oligonucleotides.

[0091]It will be understood by the skilled person that numerous nucleotide
sequences can encode the same polypeptide as a result of the degeneracy
of the genetic code.

[0092]As used herein, the term "nucleotide sequence" refers to nucleotide
sequences, oligonucleotide sequences, polynucleotide sequences and
variants, homologues, fragments and derivatives thereof (such as portions
thereof). The nucleotide sequence may be DNA or RNA of genomic or
synthetic or recombinant origin which may be double-stranded or
single-stranded whether representing the sense or antisense strand or
combinations thereof. The term nucleotide sequence may be prepared by use
of recombinant DNA techniques (for example, recombinant DNA).

[0093]Preferably, the term "nucleotide sequence" means DNA.

[0094]The terms "variant", "homologue", "derivative" or "fragment" in
relation to the present invention include any substitution of, variation
of, modification of, replacement of, deletion of or addition of one (or
more) nucleic acids from or to the sequence of a BACH nucleotide
sequence. Unless the context admits otherwise, references to "BACH" and
"BACH GPCR" include references to such variants, homologues, derivatives
and fragments of BACH.

[0095]Preferably, the resultant nucleotide sequence encodes a polypeptide
having GPCR activity, preferably having at least the same activity of the
GPCR shown as SEQ ID NO: 3 or SEQ ID NO: 5. Preferably, the term
"homologue" is intended to cover identity with respect to structure
and/or function such that the resultant nucleotide sequence encodes a
polypeptide which has GPCR activity. With respect to sequence identity
(i.e. similarity), preferably there is at least 70%, more preferably at
least 75%, more preferably at least 85%, more preferably at least 90%
sequence identity. More preferably there is at least 95%, more preferably
at least 98%, sequence identity. These terms also encompass allelic
variations of the sequences.

BACH Control Regions

[0096]For some purposes, it may be necessary to utilise or investigate
control regions of BACH. Such control regions include promoters,
enhancers and locus control regions. By a control region we mean a
nucleic acid sequence or structure which is capable of modulating the
expression of a coding sequence which is operatively linked to it.

[0097]For example, control regions are useful in generating transgenic
animals expressing BACH. Furthermore, control regions may be used to
generate expression constructs for BACH. Control regions from different
individuals in a population may be sequenced to identify non-coding
polymorphisms, which may affect the expression level of BACH. This is
described in further detail below.

[0098]Identification of control regions of BACH is straightforward, and
may be carried out in a number of ways. For example, the coding sequence
of BACH may be obtained from an organism, by screening a cDNA library
using a human or mouse BACH cDNA sequence as a probe. 5' sequences may be
obtained by screening an appropriate genomic library, or by primer
extension as known in the art. Database searching of genome databases may
also be employed. Such 5' sequences which are particularly of interest
include non-coding regions. The 5' regions may be examined by eye, or
with the aid of computer programs, to identify sequence motifs which
indicate the presence of promoter and/or enhancer regions.

[0099]Furthermore, sequence alignments may be conducted of BACH nucleic
acid sequences from two or more organisms. By aligning BACH GPCR
sequences from different species, it is possible to determine which
regions of the amino acid sequence are conserved between different
species. Such conserved regions are likely to contain control regions for
the gene in question (i.e., BACH). The mouse and human genomic sequences
as disclosed here, for example, a mouse BACH genomic sequence (SEQ ID NO:
10), may be employed for this purpose. Furthermore, BACH homologues from
other organisms may be obtained using standard methods of screening using
appropriate probes generated from the mouse and human BACH sequences. The
genome of the pufferfish (Takifugu rubripes) may also be screened to
identify a BACH homologue; comparison of the 5' non-coding region of the
Fugu BACH gene with a mouse or human genomic BACH sequence (e.g., SEQ ID
NO: 10, mouse BACH genomic sequence) may be used to identify conserved
regions containing control regions.

[0100]Deletion studies may also be conducted to identify promoter and/or
enhancer regions for BACH.

[0101]The identity of putative control regions may be confirmed by
molecular biology experiments, in which the candidate sequences are
linked to a reporter gene and the expression of the reporter detected.

BACH GPCR Associated Diseases

[0102]According to the methods and compositions described here, BACH GPCR
is useful for treating and diagnosing a range of diseases.

[0103]We demonstrate here that human BACH maps to Homo sapiens chromosome
12p13.3. Accordingly, in a specific embodiment, BACH, and agents which
bind to, agonise or antagonise BACH may be used to treat or diagnose a
disease which maps to this locus, chromosomal band, region, arm or the
same chromosome.

[0104]Furthermore, we demonstrate an expression pattern of BACH in
transgenic mice, and identify the phenotypes of such mice. These indicate
a role for BACH in sensing pain, maintaining balance and in secretion.
BACH and agents which bind to, agonise or antagonise BACH may be used to
treat or diagnose a disease in which there is a disorder in any of these.

[0105]Accordingly, in one embodiment of the invention, BACH GPCR may be
used to diagnose or treat, by any means as described in this document,
trigeminal neuralgia, orofacial pain, pain associated with toothache,
irritable bowel syndrome, Barrett's oesophagus, glaucoma, pain associated
with cancer, diabetic neuropathies, Herpes infections, HIV infections,
migraine and skin sensitivity associated with migraine, allodynia,
toothache, neuroma (whether caused by amputation, nerve transaction or
trauma), nerve compression (caused by tumours, entrapment or crush), pain
due to damage of the spinal cord or brain.

[0106]In another embodiment, BACH GPCR may be used to diagnose or treat,
by any means as described in this document, dementia, dyslexia,
dyskinesias, tremor, Parkinson's, benign essential tremor, chorea,
epilepsy or ballismus, for example occurring through stroke, trauma,
degeneration or malignancy.

[0107]In a further embodiment, BACH GPCR may be used to diagnose or treat,
by any means as described in this document, dry-eye disorders, cystic
fibrosis, hyperactive bladder, hypercholesterolaemia, dislipdaemias and
obesity.

[0108]As noted above, BACH GPCR may be used to diagnose and/or treat any
of these specific diseases ("BACH associated diseases") using any of the
methods and compositions described here.

[0109]In particular, we specifically envisage the use of nucleic acids,
vectors comprising BACH GPCR nucleic acids, polypeptides, including
homologues, variants or derivatives thereof, pharmaceutical compositions,
host cells, and transgenic animals comprising BACH GPCR nucleic acids
and/or polypeptides, for the treatment or diagnosis of the specific
diseases listed above. Furthermore, we envisage the use of compounds
capable of interacting with or binding to BACH GPCR, preferably
antagonists of a BACH GPCR, preferably a compound capable of lowering the
endogenous level of cyclic AMP in a cell, antibodies BACH BACH GPCR, as
well as methods of making or identifying these, in diagnosis or treatment
of the specific diseases mentioned above. In particular, we include the
use of any of these compounds, compositions, molecules, etc, in the
production of vaccines for treatment or prevention of the specific
diseases. We also disclose diagnostic kits for the detection of the
specific diseases in an individual.

[0110]Methods of linkage mapping to identify such or further specific
diseases treatable or diagnosable by use of BACH GPCR are known in the
art, and are also described elsewhere in this document.

[0112]As shown in the Examples and Figures, BACH is expressed in the
nervous system of mice. Altogether, the presence of LacZ staining in the
Dorsal root ganglia, the spinal cord and the trigeminal ganglion and
trigeminal nucleus indicates a very strong potential role for BACH in
pain and sensitivity (Julius and Basbaum, 20011).

[0113]This is consistent with the lower response of the BACH mutant to the
paw-pressure test the hypoalgesic responses seen in other tests such as
the tail flick test. Similarly, the expression in the viscera such as in
the tubes of the digestive system (oesphagus, gut, stomach) and sacs such
as the bladder and gall bladder, lungs, salivary glands and eyes is
strongly reminiscent of the expression pattern of P2X3 which is involved
in "stretching" or visceral pain sensitivity in these organs and as well
as neuropathic/inflammatory pain (Burnstock 2001, Cockayne et al 2000,
Souslova et al, 2000).

[0114]Examination of the expression pattern and analgesia phenotypic data
of the mutant mice lacking BACH therefore show that BACH is therefore a
target for potential treatment of trigeminal neuralgia as well as
migraine. Accordingly, therapeutic agents developed using the methods and
compositions described here may be used as analgesics. Such therapeutic
agents may comprise agonists or antagonists of BACH, preferably
antagonists of BACH.

[0115]The agents identified may be used in the treatment and management of
neuropathic, inflammatory and visceral pain. These analgesic type
therapeutics may among other conditions be used to treat Trigeminal
neuralgia, orofacial pain, pain associated with toothache, irritable
bowel syndrome, Barrett's oesophagus, glaucoma, pain associated with
cancer, diabetic neuropathies, Herpes infections, HIV infections,
migraine and skin sensitivity associated with migraine, allodynia,
toothache, neuroma (whether caused by amputation, nerve transaction or
trauma), nerve compression (caused by tumours, entrapment or crush) and
pain due to damage of the spinal cord or brain.

[0116]Orofacial pain is a consequence of trigeminal neuralgia, in which
paroxysmal pain radiates over one, or two divisions of the trigeminal
nerve. The opthalmic division is rarely affected. Drug treatment is
usually effective but if it fails surgical treatment is used. None of
these surgical treatments has proved satisfactory. No specific drug has
been developed yet.

[0117]Skin sensitivity appears among the majority of migraine sufferers.
Burstein et al published a study showing that 79 percent of 44 migraine
patients had extreme skin sensitivity. Burstein describes the extreme
effects of migraine sufferers are unable to undertake day to day tasks
such as brushing hair, wearrings or eyeglasses, or shaving beards because
of the extreme pain.

[0118]In migraine series of neuronal clusters--in the sensory ganglions,
the brainstem and the thalamus--become sensitised in a kind of domino
effect. If the sensitised cluster, a group of nerve cells that acts like
the hub of a computer network, happens to be connected to the skin, the
result can be skin sensitivity. The problem starts with the release of
inflammatory substances from the dura, and from blood vessels and nerve
endings in the brain. This oversensitizes the trigeminal ganglion. When
oversensitized, the ganglion interprets normal pressure inside the skull
as the throbbing pain of migraine. Because the trigeminal ganglion seems
to cause the primary pain of migraine, it is the target of current
migraine drugs, which block serotonin receptors in sensory neurons
connected to the dura. The drugs are often effective, but only if taken
immediately after the headache begins.

[0119]The oversensitised trigeminal ganglion may, in turn, send signals to
the nucleus caudalis, at the top of the spinal cord. Unlike the
trigeminal ganglion, this group of nerves is connected to the skin,
particularly near the eye, where the most dramatic skin sensitivity is
found in migraine sufferers. In rats, once the trigeminal ganglion has
activated the nucleus caudalis for an hour, the nucleus caudalis remains
overwrought even if the trigeminal ganglion is calmed by drugs--as
existing migraine treatments often do. The experiment also indicates that
hyper-sensitive neurons in the nucleus caudalis interpret soft touches on
the skin as pain. Although current migraine drugs often work if taken
quickly after the headache's onset, that is impossible for people who
don't have drugs handy or get the headaches while asleep. According to
Burnstein, this could explain why current anti-migraine therapies, which
work on the primary cluster, are only effective if taken during the
firsts hour after an attack has begun. An important target therefore
comprises secondary neurons.

[0121]According to an embodiment of the invention, BACH is involved in
control of balance. As shown in the Examples, BACH is expressed in
cranial nerve number 8 (vestibulocochlear nerve) and the cerebellum. BACH
negative mice are seen to have disorders in balance and motion.
Accordingly, the methods and compositions described here may be used to
identify agonists and antagonists of BACH which may be used to treat
movement disorders, motion related disorders and disorders of balance.

[0122]Furthermore, the methods and compositions described here may be used
to identify agonists and antagonists of BACH which may be used in the
treatment and management of dementia related disorders. Such disorders
include dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign
essential tremor, chorea, epilepsy and ballismus, for example occurring
through stroke, trauma, degeneration or malignancy.

[0123]Other diseases that BACH may be implicated in as derived from the
results may be balance, tremor, epilepsy all of which can be caused by
defects in genes expressed the cerebellum.

[0124]Dyslexia has been shown to have abnormal cerebellar processing
(Nicolson et al 19991) that causes (among other factors) dyslexic
patients to have a cerebellar deficit that adversely affects learning of
new skills and the performance of autonomic, overlearned skills. In other
areas of the brain purines have been shown to increase dopamine levels
and thereby enhance reward behaviour. Purinoceptors are generally
excitatory but have been shown to inhibit the release of the main
excitatory neurotransmitter in the CNS, glutamate (Mendoza-Fernandez et
al. 20001).

Secretion Related Disorders

[0125]Therapeutic agents developed to BACH may be used in the treatment
and management of dry-eye disorders, cystic fibrosis, hyperactive
bladder, hypercholesterolaemia, dislipdaemias and obesity.

Calculation of Sequence Homology

[0126]Sequence identity with respect to any of the sequences presented
here can be determined by a simple "eyeball" comparison (i.e. a strict
comparison) of any one or more of the sequences with another sequence to
see if that other sequence has, for example, at least 70% sequence
identity to the sequence(s).

[0127]Relative sequence identity can also be determined by commercially
available computer programs that can calculate % identity between two or
more sequences using any suitable algorithm for determining identity,
using for example default parameters. A typical example of such a
computer program is CLUSTAL. Other computer program methods to determine
identify and similarity between the two sequences include but are not
limited to the GCG program package (Devereux et al 1984 Nucleic Acids
Research 12: 387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).

[0128]% homology may be calculated over contiguous sequences, i.e. one
sequence is aligned with the other sequence and each amino acid in one
sequence is directly compared with the corresponding amino acid in the
other sequence, one residue at a time. This is called an "ungapped"
alignment. Typically, such ungapped alignments are performed only over a
relatively short number of residues.

[0129]Although this is a very simple and consistent method, it fails to
take into consideration that, for example, in an otherwise identical pair
of sequences, one insertion or deletion will cause the following amino
acid residues to be put out of alignment, thus potentially resulting in a
large reduction in % homology when a global alignment is performed.
Consequently, most sequence comparison methods are designed to produce
optimal alignments that take into consideration possible insertions and
deletions without penalising unduly the overall homology score. This is
achieved by inserting "gaps" in the sequence alignment to try to maximise
local homology.

[0130]However, these more complex methods assign "gap penalties" to each
gap that occurs in the alignment so that, for the same number of
identical amino acids, a sequence alignment with as few gaps as
possible--reflecting higher relatedness between the two compared
sequences--will achieve a higher score than one with many gaps. "Affine
gap costs" are typically used that charge a relatively high cost for the
existence of a gap and a smaller penalty for each subsequent residue in
the gap. This is the most commonly used gap scoring system. High gap
penalties will of course produce optimised alignments with fewer gaps.
Most alignment programs allow the gap penalties to be modified. However,
it is preferred to use the default values when using such software for
sequence comparisons. For example, when using the GCG Wisconsin Bestfit
package the default gap penalty for amino acid sequences is -12 for a gap
and -4 for each extension.

[0131]Calculation of maximum % homology therefore firstly requires the
production of an optimal alignment, taking into consideration gap
penalties. A suitable computer program for carrying out such an alignment
is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.;
Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other
software than can perform sequence comparisons include, but are not
limited to, the BLAST package (Ausubel et al., 1999 ibid--Chapter 18),
FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS
suite of comparison tools. Both BLAST and FASTA are available for offline
and online searching (Ausubel et al., 1999 ibid, pages 7-58 to 7-60).

[0132]Although the final % homology can be measured in terms of identity,
the alignment process itself is typically not based on an all-or-nothing
pair comparison. Instead, a scaled similarity score matrix is generally
used that assigns scores to each pairwise comparison based on chemical
similarity or evolutionary distance. An example of such a matrix commonly
used is the BLOSUM62 matrix--the default matrix for the BLAST suite of
programs. GCG Wisconsin programs generally use either the public default
values or a custom symbol comparison table if supplied. It is preferred
to use the public default values for the GCG package, or in the case of
other software, the default matrix, such as BLOSUM62.

[0133]Advantageously, the BLAST algorithm is employed, with parameters set
to default values. The BLAST algorithm is described in detail at
http://www.ncbi.nih gov/BLAST/blast_help.html, which is incorporated
herein by reference. The search parameters are defined as follows, can be
advantageously set to the defined default parameters.

[0134]Advantageously, "substantial identity" when assessed by BLAST
equates to sequences which match with an EXPECT value of at least about
7, preferably at least about 9 and most preferably 10 or more. The
default threshold for EXPECT in BLAST searching is usually 10.

[0135]BLAST (Basic Local Alignment Search Tool) is the heuristic search
algorithm employed by the programs blastp, blastn, blastx, tblastn, and
tblastx; these programs ascribe significance to their findings using the
statistical methods of Karlin and Altschul (Karlin and Altschul 1990,
Proc. Natl. Acad. Sci. USA 87:2264-68; Karlin and Altschul, 1993, Proc.
Natl. Acad. Sci. USA 90:5873-7; see http://www.ncbi.nih
gov/BLAST/blast_help.html) with a few enhancements. The BLAST programs
are tailored for sequence similarity searching, for example to identify
homologues to a query sequence. For a discussion of basic issues in
similarity searching of sequence databases, see Altschul et al (1994)
Nature Genetics 6:119-129.

[0136]The five BLAST programs available at http://www.ncbi.nlm nih gov
perform the following tasks: blastp--compares an amino acid query
sequence against a protein sequence database; blastn--compares a
nucleotide query sequence against a nucleotide sequence database;
blastx--compares the six-frame conceptual translation products of a
nucleotide query sequence (both strands) against a protein sequence
database; tblastn--compares a protein query sequence against a nucleotide
sequence database dynamically translated in all six reading frames (both
strands); tblastx--compares the six-frame translations of a nucleotide
query sequence against the six-frame translations of a nucleotide
sequence database.

[0137]BLAST uses the following search parameters:

[0138]HISTOGRAM--Display a histogram of scores for each search; default is
yes. (See parameter H in the BLAST Manual).

[0139]DESCRIPTIONS--Restricts the number of short descriptions of matching
sequences reported to the number specified; default limit is 100
descriptions. (See parameter V in the manual page).

[0140]EXPECT--The statistical significance threshold for reporting matches
against database sequences; the default value is 10, such that 10 matches
are expected to be found merely by chance, according to the stochastic
model of Karlin and Altschul (1990). If the statistical significance
ascribed to a match is greater than the EXPECT threshold, the match will
not be reported. Lower EXPECT thresholds are more stringent, leading to
fewer chance matches being reported. Fractional values are acceptable.
(See parameter E in the BLAST Manual).

[0141]CUTOFF--Cutoff score for reporting high-scoring segment pairs. The
default value is calculated from the EXPECT value (see above). HSPs are
reported for a database sequence only if the statistical significance
ascribed to them is at least as high as would be ascribed to a lone HSP
having a score equal to the CUTOFF value. Higher CUTOFF values are more
stringent, leading to fewer chance matches being reported. (See parameter
S in the BLAST Manual). Typically, significance thresholds can be more
intuitively managed using EXPECT.

[0142]ALIGNMENTS--Restricts database sequences to the number specified for
which high-scoring segment pairs (HSPs) are reported; the default limit
is 50. If more database sequences than this happen to satisfy the
statistical significance threshold for reporting (see EXPECT and CUTOFF
below), only the matches ascribed the greatest statistical significance
are reported. (See parameter B in the BLAST Manual).

[0144]STRAND--Restrict a TBLASTN search to just the top or bottom strand
of the database sequences; or restrict a BLASTN, BLASTX or TBLASTX search
to just reading frames on the top or bottom strand of the query sequence.

[0145]FILTER--Mask off segments of the query sequence that have low
compositional complexity, as determined by the SEG program of Wootton &
Federhen (1993) Computers and Chemistry 17:149-163, or segments
consisting of short-periodicity internal repeats, as determined by the
XNU program of Claverie & States (1993) Computers and Chemistry
17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman
(see http://www.ncbi.nlm nih gov). Filtering can eliminate statistically
significant but biologically uninteresting reports from the blast output
(e.g., hits against common acidic-, basic- or proline-rich regions),
leaving the more biologically interesting regions of the query sequence
available for specific matching against database sequences.

[0146]Low complexity sequence found by a filter program is substituted
using the letter "N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") and
the letter "X" in protein sequences (e.g., "XXXXXXXXX").

[0147]Filtering is only applied to the query sequence (or its translation
products), not to database sequences. Default filtering is DUST for
BLASTN, SEG for other programs.

[0148]It is not unusual for nothing at all to be masked by SEG, XNU, or
both, when applied to sequences in SWISS-PROT, so filtering should not be
expected to always yield an effect. Furthermore, in some cases, sequences
are masked in their entirety, indicating that the statistical
significance of any matches reported against the unfiltered query
sequence should be suspect.

[0149]NCBI-gi--Causes NCBI gi identifiers to be shown in the output, in
addition to the accession and/or locus name.

[0150]Most preferably, sequence comparisons are conducted using the simple
BLAST search algorithm provided at http://www.ncbi.nlm nih gov/BLAST. In
some embodiments of the present invention, no gap penalties are used when
determining sequence identity.

Hybridization

[0151]The present invention also encompasses nucleotide sequences that are
capable of hybridising to the sequences presented herein, or any fragment
or derivative thereof, or to the complement of any of the above.

[0152]Hybridization means a "process by which a strand of nucleic acid
joins with a complementary strand through base pairing" (Coombs J (1994)
Dictionary of Biotechnology, Stockton Press, New York N.Y.) as well as
the process of amplification as carried out in polymerase chain reaction
technologies as described in Dieffenbach C W and G S Dveksler (1995, PCR
Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.).

[0153]Hybridization conditions are based on the melting temperature (Tm)
of the nucleic acid binding complex, as taught in Berger and Kimmel
(1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol
152, Academic Press, San Diego CA), and confer a defined "stringency" as
explained below.

[0154]Nucleotide sequences of the invention capable of selectively
hybridising to the nucleotide sequences presented herein, or to their
complement, will be generally at least 70%, preferably at least 75%, more
preferably at least 85 or 90% and even more preferably at least 95% or
98% homologous to the corresponding nucleotide sequences presented herein
over a region of at least 20, preferably at least 25 or 30, for instance
at least 40, 60 or 100 or more contiguous nucleotides. Preferred
nucleotide sequences of the invention will comprise regions homologous to
SEQ ID NO: 1, 2, 4 or 10, preferably at least 70%, 80% or 90% and more
preferably at least 95% homologous to one of the sequences.

[0155]The term "selectively hybridizable" means that the nucleotide
sequence used as a probe is used under conditions where a target
nucleotide sequence of the invention is found to hybridize to the probe
at a level significantly above background. The background hybridization
may occur because of other nucleotide sequences present, for example, in
the cDNA or genomic DNA library being screened. In this event, background
implies a level of signal generated by interaction between the probe and
a non-specific DNA member of the library which is less than 10 fold,
preferably less than 100 fold as intense as the specific interaction
observed with the target DNA. The intensity of interaction may be
measured, for example, by radiolabelling the probe, e.g. with 32P.

[0156]Also included within the scope of the present invention are
nucleotide sequences that are capable of hybridizing to the nucleotide
sequences presented herein under conditions of intermediate to maximal
stringency. Hybridization conditions are based on the melting temperature
(Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel
(1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol
152, Academic Press, San Diego Calif.), and confer a defined "stringency"
as explained below.

[0157]Maximum stringency typically occurs at about Tm-5° C.
(5° C. below the Tm of the probe); high stringency at about
5° C. to 10° C. below Tm; intermediate stringency at about
10° C. to 20° C. below Tm; and low stringency at about
20° C. to 25° C. below Tm. As will be understood by those
of skill in the art, a maximum stringency hybridization can be used to
identify or detect identical nucleotide sequences while an intermediate
(or low) stringency hybridization can be used to identify or detect
similar or related nucleotide sequences.

[0158]In a preferred embodiment, the present invention covers nucleotide
sequences that can hybridise to one or more of the BACH GPCR nucleotide
sequences of the present invention under stringent conditions (e.g.
65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M
Na3 Citrate pH 7.0). Where the nucleotide sequence of the invention
is double-stranded, both strands of the duplex, either individually or in
combination, are encompassed by the present invention. Where the
nucleotide sequence is single-stranded, it is to be understood that the
complementary sequence of that nucleotide sequence is also included
within the scope of the present invention.

[0159]The present invention also encompasses nucleotide sequences that are
capable of hybridising to the sequences that are complementary to the
sequences presented herein, or any fragment or derivative thereof.
Likewise, the present invention encompasses nucleotide sequences that are
complementary to sequences that are capable of hybridising to the
sequence of the present invention. These types of nucleotide sequences
are examples of variant nucleotide sequences. In this respect, the term
"variant" encompasses sequences that are complementary to sequences that
are capable of hydridising to the nucleotide sequences presented herein.
Preferably, however, the term "variant" encompasses sequences that are
complementary to sequences that are capable of hydridising under
stringent conditions (eg. 65° C. and 0.1×SSC {
1×SSC=0.15 M NaCl, 0.015 Na3 citrate pH 7.0}) to the
nucleotide sequences presented herein.

Cloning of BACH GPCR and Homologues

[0160]The present invention also encompasses nucleotide sequences that are
complementary to the sequences presented here, or any fragment or
derivative thereof. If the sequence is complementary to a fragment
thereof then that sequence can be used as a probe to identify and clone
similar GPCR sequences in other organisms etc.

[0161]The present invention thus enables the cloning of BACH GPCR, its
homologues and other structurally or functionally related genes from
human and other species such as mouse, pig, sheep, etc to be
accomplished. Polynucleotides of the invention, which are identical or
sufficiently identical to a nucleotide sequence contained in SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 4 or a mouse BACH genomic sequence (SEQ ID
NO: 10), or a fragment thereof, may be used as hybridization probes for
cDNA and genomic DNA as appropriate, to isolate partial or full-length
cDNAs and genomic clones encoding BACH GPCR from appropriate libraries.
Such probes may also be used to isolate cDNA and genomic clones of other
genes (including genes encoding homologues and orthologues from species
other than human) that have sequence similarity, preferably high sequence
similarity, to the BACH GPCR gene. Hybridization screening, cloning and
sequencing techniques are known to those of skill in the art and are
described in, for example, Sambrook et al (supra).

[0162]Typically nucleotide sequences suitable for use as probes are 70%
identical, preferably 80% identical, more preferably 90% identical, even
more preferably 95% identical to that of the referent. The probes
generally will comprise at least 15 nucleotides. Preferably, such probes
will have at least 30 nucleotides and may have at least 50 nucleotides.
Particularly preferred probes will range between 150 and 500 nucleotides,
more particularly about 300 nucleotides.

[0163]In one embodiment, to obtain a polynucleotide encoding a BACH GPCR
polypeptide, including homologues and orthologues from species other than
human, comprises the steps of screening an appropriate library under
stringent hybridization conditions with a labelled probe having the SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or a mouse BACH genomic sequence
(SEQ ID NO: 10), or a fragment thereof and isolating partial or
full-length cDNA and genomic clones containing said polynucleotide
sequence. Such hybridization techniques are well known to those of skill
in the art. Stringent hybridization conditions are as defined above or
alternatively conditions under overnight incubation at 42 degrees C. in a
solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM
trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's
solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared
salmon sperm DNA, followed by washing the filters in 0.1×SSC at
about 65 degrees C.

[0164]Functional Assay for BACH GPCR

[0165]The cloned putative BACH GPCR polynucleotides may be verified by
sequence analysis or functional assays. For example, the putative BACH
GPCR or homologue may be assayed for receptor activity as follows. Capped
RNA transcripts from linearized plasmid templates encoding the BACH
receptor cDNAs of the invention are synthesized in vitro with RNA
polymerases in accordance with standard procedures. In vitro transcripts
are suspended in water at a final concentration of 0.2 mg/ml. Ovarian
lobes are removed from adult female toads, Stage V defolliculated oocytes
are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl
bolus using a microinjection apparatus. Two electrode voltage clamps are
used to measure the currents from individual Xenopus oocytes in response
to agonist exposure. Recordings are made in Ca2+ free Barth's medium
at room temperature. The Xenopus system may also be used to screen known
ligands and tissue/cell extracts for activating ligands, as described in
further detail below.

Expression Assays for BACH GPCR

[0166]In order to design useful therapeutics for treating BACH GPCR
associated diseases, it is useful to determine the expression profile of
BACH (whether wild-type or a particular mutant). Thus, methods known in
the art may be used to determine the organs, tissues and cell types (as
well as the developmental stages) in which BACH is expressed. For
example, traditional or "electronic" Northerns may be conducted.
Reverse-transcriptase PCR (RT-PCR) may also be employed to assay
expression of the BACH gene or mutant. More sensitive methods for
determining the expression profile of BACH include RNAse protection
assays, as known in the art.

[0167]Northern analysis is a laboratory technique used to detect the
presence of a transcript of a gene and involves the hybridization of a
labeled nucleotide sequence to a membrane on which RNAs from a particular
cell type or tissue have been bound. (Sambrook, supra, ch. 7 and Ausubel,
F. M. et al. supra, ch. 4 and 16.) Analogous computer techniques
("electronic Northerns") applying BLAST may be used to search for
identical or related molecules in nucleotide databases such as GenBank or
the LIFESEQ database (Incyte Pharmaceuticals). This type of analysis has
advantages in that they may be faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search can
be modified to determine whether any particular match is categorized as
exact or homologous.

[0168]The polynucleotides and polypeptides of the present invention,
including the probes described above, may be employed as research
reagents and materials for discovery of treatments and diagnostics to
animal and human disease, as explained in further detail elsewhere in
this document.

Expression of BACH GPCR Polypeptides

[0169]In order to express a biologically active BACH GPCR, the nucleotide
sequences encoding BACH GPCR or homologues, variants, or derivatives
thereof are inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence.

[0170]Methods which are well known to those skilled in the art are used to
construct expression vectors containing sequences encoding BACH GPCR and
appropriate transcriptional and translational control elements. These
methods include in vitro recombinant DNA techniques, synthetic
techniques, and in vivo genetic recombination. Such techniques are
described in Sambrook, J. et al. (1989; Molecular Cloning, A Laboratory
Manual, ch. 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, N.Y.)
and Ausubel, F. M. et al. (1995 and periodic supplements; Current
Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New
York, N.Y.).

[0172]The "control elements" or "regulatory sequences" are those
non-translated regions of the vector (i.e., enhancers, promoters, and 5'
and 3' untranslated regions) which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary in
their strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used. For example,
when cloning in bacterial systems, inducible promoters such as the hybrid
lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.)
or PSPORT1 plasmid (GIBCO/BRL), and the like, may be used. The
baculovirus polyhedrin promoter may be used in insect cells. Promoters or
enhancers derived from the genomes of plant cells (e.g., heat shock,
RUBISCO, and storage protein genes) or from plant viruses (e.g., viral
promoters or leader sequences) may be cloned into the vector. In
mammalian cell systems, promoters from mammalian genes or from mammalian
viruses are preferable. If it is necessary to generate a cell line that
contains multiple copies of the sequence encoding BACH GPCR, vectors
based on SV40 or EBV may be used with an appropriate selectable marker.

[0173]In bacterial systems, a number of expression vectors may be selected
depending upon the use intended for BACH GPCR. For example, when large
quantities of BACH GPCR are needed for the induction of antibodies,
vectors which direct high level expression of fusion proteins that are
readily purified may be used. Such vectors include, but are not limited
to, multifunctional E. coli cloning and expression vectors such as
BLUESCRIPT (Stratagene), in which the sequence encoding BACH GPCR may be
ligated into the vector in frame with sequences for the amino-terminal
Met and the subsequent 7 residues of β-galactosidase so that a
hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M. Schuster
(1989) J. Biol. Chem. 264:5503-5509), and the like. pGEX vectors
(Promega, Madison, Wis.) may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed cells
by adsorption to glutathione-agarose beads followed by elution in the
presence of free glutathione. Proteins made in such systems may be
designed to include heparin, thrombin, or factor XA protease cleavage
sites so that the cloned polypeptide of interest can be released from the
GST moiety at will.

[0174]In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha factor,
alcohol oxidase, and PGH, may be used. For reviews, see Ausubel (supra)
and Grant et al. (1987; Methods Enzymol. 153:516-544).

[0175]In cases where plant expression vectors are used, the expression of
sequences encoding BACH GPCR may be driven by any of a number of
promoters. For example, viral promoters such as the 35S and 19S promoters
of CaMV may be used alone or in combination with the omega leader
sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)
Alternatively, plant promoters such as the small subunit of RUBISCO or
heat shock promoters may be used. (Coruzzi, G. et al. (1984) EMBO J.
3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,
J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs
can be introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. Such techniques are described in a number
of generally available reviews. (See, for example, Hobbs, S. or Murry, L.
E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill,
New York, N.Y.; pp. 191-196.).

[0176]An insect system may also be used to express BACH GPCR. For example,
in one such system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes in Spodoptera
frugiperda cells or in Trichoplusia larvae. The sequences encoding BACH
GPCR may be cloned into a non-essential region of the virus, such as the
polyhedrin gene, and placed under control of the polyhedrin promoter.
Successful insertion of BACH GPCR will render the polyhedrin gene
inactive and produce recombinant virus lacking coat protein. The
recombinant viruses may then be used to infect, for example, S.
frugiperda cells or Trichoplusia larvae in which BACH GPCR may be
expressed. (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.
91:3224-3227.)

[0177]In mammalian host cells, a number of viral-based expression systems
may be utilized. In cases where an adenovirus is used as an expression
vector, sequences encoding BACH GPCR may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter and
tripartite leader sequence. Insertion in a non-essential E1 or E3 region
of the viral genome may be used to obtain a viable virus which is capable
of expressing BACH GPCR in infected host cells. (Logan, J. and T. Shenk
(1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription
enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.

[0178]Thus, for example, the BACH receptors of the present invention are
expressed in either human embryonic kidney 293 (HEK293) cells or adherent
dhfr CHO cells. To maximize receptor expression, typically all 5' and 3'
untranslated regions (UTRs) are removed from the receptor cDNA prior to
insertion into a pCDN or pCDNA3 vector. The cells are transfected with
individual receptor cDNAs by lipofectin and selected in the presence of
400 mg/ml G418. After 3 weeks of selection, individual clones are picked
and expanded for further analysis. HEK293 or CHO cells transfected with
the vector alone serve as negative controls. To isolate cell lines stably
expressing the individual receptors, about 24 clones are typically
selected and analyzed by Northern blot analysis. Receptor mRNAs are
generally detectable in about 50% of the G418-resistant clones analyzed.

[0179]Human artificial chromosomes (HACs) may also be employed to deliver
larger fragments of DNA than can be contained and expressed in a plasmid.
HACs of about 6 kb to 10 Mb are constructed and delivered via
conventional delivery methods (liposomes, polycationic amino polymers, or
vesicles) for therapeutic purposes.

[0180]Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding BACH GPCR. Such signals
include the ATG initiation codon and adjacent sequences. In cases where
sequences encoding BACH GPCR and its initiation codon and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals including
the ATG initiation codon should be provided. Furthermore, the initiation
codon should be in the correct reading frame to ensure translation of the
entire insert. Exogenous translational elements and initiation codons may
be of various origins, both natural and synthetic. The efficiency of
expression may be enhanced by the inclusion of enhancers appropriate for
the particular cell system used, such as those described in the
literature. (Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)

[0181]In addition, a host cell strain may be chosen for its ability to
modulate expression of the inserted sequences or to process the expressed
protein in the desired fashion. Such modifications of the polypeptide
include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation, lipidation, and acylation.
Post-translational processing which cleaves a "prepro" form of the
protein may also be used to facilitate correct insertion, folding, and/or
function. Different host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g., CHO,
HeLa, MDCK, HEK293, and WI38), are available from the American Type
Culture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure the
correct modification and processing of the foreign protein.

[0182]For long term, high yield production of recombinant proteins, stable
expression is preferred. For example, cell lines capable of stably
expressing BACH GPCR can be transformed using expression vectors which
may contain viral origins of replication and/or endogenous expression
elements and a selectable marker gene on the same or on a separate
vector. Following the introduction of the vector, cells may be allowed to
grow for about 1 to 2 days in enriched media before being switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and recovery of
cells which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.

[0183]Any number of selection systems may be used to recover transformed
cell lines. These include, but are not limited to, the herpes simplex
virus thymidine kinase genes (Wigler, M. et al. (1977) Cell 11:223-32)
and adenine phosphoribosyltransferase genes (Lowy, I. et al. (1980) Cell
22:817-23), which can be employed in tk.sup.- or apr.sup.- cells,
respectively. Also, antimetabolite, antibiotic, or herbicide resistance
can be used as the basis for selection. For example, dhfr confers
resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.
Sci. 77:3567-70); npt confers resistance to the aminoglycosides neomycin
and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and
als or pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra). Additional selectable
genes have been described, for example, trpB, which allows cells to
utilize indole in place of tryptophan, or hisD, which allows cells to
utilize histinol in place of histidine. (Hartman, S. C. and R. C.
Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51.) Recently, the use of
visible markers has gained popularity with such markers as anthocyanins,
β-glucuronidase and its substrate GUS, and luciferase and its
substrate luciferin. These markers can be used not only to identify
transformants, but also to quantify the amount of transient or stable
protein expression attributable to a specific vector system. (Rhodes, C.
A. et al. (1995) Methods Mol. Biol. 55:121-131.)

[0184]Although the presence/absence of marker gene expression suggests
that the gene of interest is also present, the presence and expression of
the gene may need to be confirmed. For example, if the sequence encoding
BACH GPCR is inserted within a marker gene sequence, transformed cells
containing sequences encoding BACH GPCR can be identified by the absence
of marker gene function. Alternatively, a marker gene can be placed in
tandem with a sequence encoding BACH GPCR under the control of a single
promoter. Expression of the marker gene in response to induction or
selection usually indicates expression of the tandem gene as well.

[0185]Alternatively, host cells which contain the nucleic acid sequence
encoding BACH GPCR and express BACH GPCR may be identified by a variety
of procedures known to those of skill in the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and
protein bioassay or immunoassay techniques which include membrane,
solution, or chip based technologies for the detection and/or
quantification of nucleic acid or protein sequences.

[0186]The presence of polynucleotide sequences encoding BACH GPCR can be
detected by DNA-DNA or DNA-RNA hybridization or amplification using
probes or fragments or fragments of polynucleotides encoding BACH GPCR.
Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding BACH GPCR
to detect transformants containing DNA or RNA encoding BACH GPCR.

[0187]A variety of protocols for detecting and measuring the expression of
BACH GPCR, using either polyclonal or monoclonal antibodies specific for
the protein, are known in the art. Examples of such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies reactive to
two non-interfering epitopes on BACH GPCR is preferred, but a competitive
binding assay may be employed. These and other assays are well described
in the art, for example, in Hampton, R. et al. (1990; Serological
Methods, a Laboratory Manual, Section IV, APS Press, St Paul, Minn.) and
in Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

[0188]A wide variety of labels and conjugation techniques are known by
those skilled in the art and may be used in various nucleic acid and
amino acid assays. Means for producing labeled hybridization or PCR
probes for detecting sequences related to polynucleotides encoding BACH
GPCR include oligolabeling, nick translation, end-labeling, or PCR
amplification using a labeled nucleotide. Alternatively, the sequences
encoding BACH GPCR, or any fragments thereof, may be cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art,
are commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a
variety of commercially available kits, such as those provided by
Pharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S.
Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules or
labels which may be used for ease of detection include radionuclides,
enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as
substrates, cofactors, inhibitors, magnetic particles, and the like.

[0189]Host cells transformed with nucleotide sequences encoding BACH GPCR
may be cultured under conditions suitable for the expression and recovery
of the protein from cell culture. The protein produced by a transformed
cell may be located in the cell membrane, secreted or contained
intracellularly depending on the sequence and/or the vector used. As will
be understood by those of skill in the art, expression vectors containing
polynucleotides which encode BACH GPCR may be designed to contain signal
sequences which direct secretion of BACH GPCR through a prokaryotic or
eukaryotic cell membrane. Other constructions may be used to join
sequences encoding BACH GPCR to nucleotide sequences encoding a
polypeptide domain which will facilitate purification of soluble
proteins. Such purification facilitating domains include, but are not
limited to, metal chelating peptides such as histidine-tryptophan modules
that allow purification on immobilized metals, protein A domains that
allow purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp.,
Seattle, Wash.). The inclusion of cleavable linker sequences, such as
those specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.), between the purification domain and the BACH GPCR encoding
sequence may be used to facilitate purification. One such expression
vector provides for expression of a fusion protein containing BACH GPCR
and a nucleic acid encoding 6 histidine residues preceding a thioredoxin
or an enterokinase cleavage site. The histidine residues facilitate
purification on immobilized metal ion affinity chromatography (IMIAC;
described in Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281),
while the enterokinase cleavage site provides a means for purifying BACH
GPCR from the fusion protein. A discussion of vectors which contain
fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.
12:441-453).

[0190]Fragments of BACH GPCR may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the Applied
Biosystems 431A peptide synthesizer (Perkin Elmer). Various fragments of
BACH GPCR may be synthesized separately and then combined to produce the
full length molecule.

Biosensors

[0191]The BACH polypeptides, nucleic acids, probes, antibodies, expression
vectors and ligands are useful as (and for the production of) biosensors.

[0192]According to Aizawa (1988), Anal. Chem. Symp. 17: 683, a biosensor
is defined as being a unique combination of a receptor for molecular
recognition, for example a selective layer with immobilized antibodies or
receptors such as a BACH G-protein coupled receptor, and a transducer for
transmitting the values measured. One group of such biosensors will
detect the change which is caused in the optical properties of a surface
layer due to the interaction of the receptor with the surrounding medium.
Among such techniques may be mentioned especially ellipso-metry and
surface plasmon resonance. Biosensors incorporating BACH may be used to
detect the presence or level of BACH ligands, for example, nucleotides
such as purines or purine analogues, or analogues of these ligands. The
construction of such biosensors is well known in the art.

[0193]Thus, cell lines expressing BACH receptor may be used as reporter
systems for detection of ligands such as ATP via receptor-promoted
formation of [3H]inositol phosphates or other second messengers (Watt et
al., 1998, J Biol Chem May 29; 273(22):14053-8). Receptor-ligand
biosensors are also described in Hoffman et al., 2000, Proc Natl Acad Sci
USA October 10; 97(21):11215-20. Optical and other biosensors comprising
BACH may also be used to detect the level or presence of interaction with
G-proteins and other proteins, as described by, for example, Figler et
al, 1997, Biochemistry December 23; 36(51):16288-99 and Sarrio et al.,
2000, Mol Cell Biol 2000 July; 20(14):5164-74). Sensor units for
biosensors are described in, for example, U.S. Pat. No. 5,492,840.

Screening Assays

[0194]The BACH GPCR polypeptide of the present invention, including
homologues, variants, and derivatives, whether natural or recombinant,
may be employed in a screening process for compounds which bind the
receptor and which activate (agonists) or inhibit activation of
(antagonists) of BACH. Thus, polypeptides of the invention may also be
used to assess the binding of small molecule substrates and ligands in,
for example, cells, cell-free preparations, chemical libraries, and
natural product mixtures. These substrates and ligands may be natural
substrates and ligands or may be structural or functional mimetics. See
Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).

[0195]BACH GPCR polypeptides are responsible for many biological
functions, including many pathologies. Accordingly, it is desirous to
find compounds and drugs which stimulate BACH GPCR on the one hand and
which can inhibit the function of BACH GPCR on the other hand. In
general, agonists and antagonists are employed for therapeutic and
prophylactic purposes for such conditions as trigeminal neuralgia,
orofacial pain, pain associated with toothache, irritable bowel syndrome,
Barrett's oesophagus, glaucoma, pain associated with cancer, diabetic
neuropathies, Herpes infections, HIV infections, migraine and skin
sensitivity associated with migraine, allodynia, toothache, neuroma
(whether caused by amputation, nerve transaction or trauma), nerve
compression (caused by tumours, entrapment or crush), pain due to damage
of the spinal cord or brain; dementia, dyslexia, dyskinesias, tremor,
Parkinson's, benign essential tremor, chorea, epilepsy and ballismus, for
example occurring through stroke, trauma, degeneration or malignancy; or
dry-eye disorders, cystic fibrosis, hyperactive bladder,
hypercholesterolaemia, dislipdaemias and obesity.

[0196]Rational design of candidate compounds likely to be able to interact
with BACH GPCR protein may be based upon structural studies of the
molecular shapes of a polypeptide according to the invention. One means
for determining which sites interact with specific other proteins is a
physical structure determination, e.g., X-ray crystallography or
two-dimensional NMR techniques. These will provide guidance as to which
amino acid residues form molecular contact regions. For a detailed
description of protein structural determination, see, e.g., Blundell and
Johnson (1976) Protein Crystallography, Academic Press, New York.

[0197]An alternative to rational design uses a screening procedure which
involves in general producing appropriate cells which express the BACH
receptor polypeptide of the present invention on the surface thereof.
Such cells include cells from animals, yeast, Drosophila or E. coli.
Cells expressing the receptor (or cell membrane containing the expressed
receptor) are then contacted with a test compound to observe binding, or
stimulation or inhibition of a functional response. For example, Xenopus
oocytes may be injected with BACH mRNA or polypeptide, and currents
induced by exposure to test compounds measured by use of voltage clamps
measured, as described in further detail elsewhere.

[0198]Furthermore, microphysiometric assays may be employed to assay BACH
receptor activity. Activation of a wide variety of secondary messenger
systems results in extrusion of small amounts of acid from a cell. The
acid formed is largely as a result of the increased metabolic activity
required to fuel the intracellular signalling process. The pH changes in
the media surrounding the cell are very small but are detectable by, for
example, the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo
Park, Calif.). The CYTOSENSOR is thus capable of detecting the activation
of a receptor which is coupled to an energy utilizing intracellular
signaling pathway such as the G-protein coupled receptor of the present
invention.

[0199]Instead of testing each candidate compound individually with the
BACH receptor, a library or bank of candidate ligands may advantageously
be produced and screened. Thus, for example, a bank of over 200 putative
receptor ligands has been assembled for screening. The bank comprises:
transmitters, hormones and chemokines known to act via a human seven
transmembrane (7TM) receptor; naturally occurring compounds which may be
putative agonists for a human 7TM receptor, non-mammalian, biologically
active peptides for which a mammalian counterpart has not yet been
identified; and compounds not found in nature, but which activate 7TM
receptors with unknown natural ligands. This bank is used to screen the
receptor for known ligands, using both functional (i.e. calcium, cAMP,
microphysiometer, oocyte electrophysiology, etc, see elsewhere) as well
as binding assays as described in further detail elsewhere. However, a
large number of mammalian receptors exist for which there remains, as
yet, no cognate activating ligand (agonist) or deactivating ligand
(antagonist). Thus, active ligands for these receptors may not be
included within the ligands banks as identified to date. Accordingly, the
BACH receptor of the invention is also functionally screened (using
calcium, cAMP, microphysiometer, ooyte electrophysiology, etc.,
functional screens) against tissue extracts to identify natural ligands.
Extracts that produce positive functional responses can be sequentially
subfractionated, with the fractions being assayed as described here,
until an activating ligand is isolated and identified.

[0200]7TM receptors which are expressed in HEK 293 cells have been shown
to be coupled functionally to activation of PLC and calcium mobilization
and/or cAMP stimuation or inhibition. One screening technique therefore
includes the use of cells which express the BACH GPCR receptor of this
invention (for example, transfected Xenopus oocytes, CHO or HEK293 cells)
in a system which measures extracellular pH or intracellular calcium
changes caused by receptor activation. In this technique, compounds may
be contacted with cells expressing the receptor polypeptide of the
present invention. A second messenger response, e.g., signal
transduction, pH changes, or changes in calcium level, is then measured
to determine whether the potential compound activates or inhibits the
receptor.

[0201]In such experiments, basal calcium levels in the HEK 293 cells in
receptor-transfected or vector control cells are observed to be in the
normal, 100 nM to 200 nM, range. HEK 293 cells expressing BACH GPCR or
recombinant BACH GPCR are loaded with fura 2 and in a single day more
than 150 selected ligands or tissue/cell extracts are evaluated for
agonist induced calcium mobilization. Similarly, HEK 293 cells expressing
BACH GPCR or recombinant BACH GPCR are evaluated for the stimulation or
inhibition of cAMP production using standard cAMP quantitation assays.
Agonists presenting a calcium transient or cAMP fluctuation are tested in
vector control cells to determine if the response is unique to the
transfected cells expressing receptor.

[0202]Another method involves screening for receptor inhibitors by
determining inhibition or stimulation of BACH receptor-mediated cAMP
and/or adenylate cyclase accumulation. Such a method involves
transfecting a eukaryotic cell with the receptor of this invention to
express the receptor on the cell surface. The cell is then exposed to
potential antagonists in the presence of the receptor of this invention.
The amount of cAMP accumulation is then measured. If the potential
antagonist binds the receptor, and thus inhibits receptor binding, the
levels of receptor-mediated cAMP, or adenylate cyclase, activity will be
reduced or increased.

[0203]Another method for detecting agonists or antagonists for the
receptor of the present invention is the yeast based technology as
described in U.S. Pat. No. 5,482,835, incorporated by reference herein.

[0204]Where the candidate compounds are proteins, in particular antibodies
or peptides, libraries of candidate compounds may be screened using phage
display techniques. Phage display is a protocol of molecular screening
which utilises recombinant bacteriophage. The technology involves
transforming bacteriophage with a gene that encodes one compound from the
library of candidate compounds, such that each phage or phagemid
expresses a particular candidate compound. The transformed bacteriophage
(which preferably is tethered to a solid support) expresses the
appropriate candidate compound and displays it on their phage coat.
Specific candidate compounds which are capable of binding to a
polypeptide or peptide of the invention are enriched by selection
strategies based on affinity interaction. The successful candidate agents
are then characterised. Phage display has advantages over standard
affinity ligand screening technologies. The phage surface displays the
candidate agent in a three dimensional configuration, more closely
resembling its naturally occurring conformation. This allows for more
specific and higher affinity binding for screening purposes.

[0205]Another method of screening a library of compounds utilises
eukaryotic or prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing a library of compounds. Such cells,
either in viable or fixed form, can be used for standard binding-partner
assays. See also Parce et al. (1989) Science 246:243-247; and Owicki et
al. (1990) Proc. Nat'l Acad. Sci. USA 87; 4007-4011, which describe
sensitive methods to detect cellular responses. Competitive assays are
particularly useful, where the cells expressing the library of compounds
are contacted or incubated with a labelled antibody known to bind to a
BACH polypeptide of the present invention, such as 125I-antibody,
and a test sample such as a candidate compound whose binding affinity to
the binding composition is being measured. The bound and free labelled
binding partners for the polypeptide are then separated to assess the
degree of binding. The amount of test sample bound is inversely
proportional to the amount of labelled antibody binding to the
polypeptide.

[0206]Any one of numerous techniques can be used to separate bound from
free binding partners to assess the degree of binding. This separation
step could typically involve a procedure such as adhesion to filters
followed by washing, adhesion to plastic following by washing, or
centrifugation of the cell membranes.

[0207]Still another approach is to use solubilized, unpurified or
solubilized purified polypeptide or peptides, for example extracted from
transformed eukaryotic or prokaryotic host cells. This allows for a
"molecular" binding assay with the advantages of increased specificity,
the ability to automate, and high drug test throughput.

[0208]Another technique for candidate compound screening involves an
approach which provides high throughput screening for new compounds
having suitable binding affinity, e.g., to a polypeptide of the
invention, and is described in detail in International Patent application
no. WO 84/03564 (Commonwealth Serum Labs.), published on Sep. 13, 1984.
First, large numbers of different small peptide test compounds are
synthesized on a solid substrate, e.g., plastic pins or some other
appropriate surface; see Fodor et al. (1991). Then all the pins are
reacted with solubilized polypeptide of the invention and washed. The
next step involves detecting bound polypeptide. Compounds which interact
specifically with the polypeptide will thus be identified.

[0209]Ligand binding assays provide a direct method for ascertaining
receptor pharmacology and are adaptable to a high throughput format. The
purified ligand for a receptor may be radiolabeled to high specific
activity (50-2000 Ci/mmol) for binding studies. A determination is then
made that the process of radiolabeling does not diminish the activity of
the ligand towards its receptor. Assay conditions for buffers, ions, pH
and other modulators such as nucleotides are optimized to establish a
workable signal to noise ratio for both membrane and whole cell receptor
sources. For these assays, specific receptor binding is defined as total
associated radioactivity minus the radioactivity measured in the presence
of an excess of unlabeled competing ligand. Where possible, more than one
competing ligand is used to define residual nonspecific binding.

[0210]The assays may simply test binding of a candidate compound wherein
adherence to the cells bearing the receptor is detected by means of a
label directly or indirectly associated with the candidate compound or in
an assay involving competition with a labeled competitor. Further, these
assays may test whether the candidate compound results in a signal
generated by activation of the receptor, using detection systems
appropriate to the cells bearing the receptor at their surfaces
Inhibitors of activation are generally assayed in the presence of a known
agonist and the effect on activation by the agonist by the presence of
the candidate compound is observed.

[0211]Further, the assays may simply comprise the steps of mixing a
candidate compound with a solution containing a BACH GPCR polypeptide to
form a mixture, measuring BACH GPCR activity in the mixture, and
comparing the BACH GPCR activity of the mixture to a standard.

[0212]The BACH GPCR cDNA, protein and antibodies to the protein may also
be used to configure assays for detecting the effect of added compounds
on the production of BACH GPCR mRNA and protein in cells. For example, an
ELISA may be constructed for measuring secreted or cell associated levels
of BACH GPCR protein using monoclonal and polyclonal antibodies by
standard methods known in the art, and this can be used to discover
agents which may inhibit or enhance the production of BACH GPCR (also
called antagonist or agonist, respectively) from suitably manipulated
cells or tissues. Standard methods for conducting screening assays are
well understood in the art.

[0213]Examples of potential BACH GPCR antagonists include antibodies or,
in some cases, nucleotides and their analogues, including purines and
purine analogues, oligonucleotides or proteins which are closely related
to the ligand of the BACH GPCR, e.g., a fragment of the ligand, or small
molecules which bind to the receptor but do not elicit a response, so
that the activity of the receptor is prevented.

[0214]The present invention therefore also provides a compound capable of
binding specifically to a BACH polypeptide and/or peptide of the present
invention.

[0215]The term "compound" refers to a chemical compound (naturally
occurring or synthesised), such as a biological macromolecule (e.g.,
nucleic acid, protein, non-peptide, or organic molecule), or an extract
made from biological materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues, or even an inorganic element
or molecule. Preferably the compound is an antibody.

[0216]The materials necessary for such screening to be conducted may be
packaged into a screening kit. Such a screening kit is useful for
identifying agonists, antagonists, ligands, receptors, substrates,
enzymes, etc. for BACH GPCR polypeptides or compounds which decrease or
enhance the production of BACH GPCR polypeptides. The screening kit
comprises: (a) a BACH GPCR polypeptide; (b) a recombinant cell expressing
a BACH GPCR polypeptide; (c) a cell membrane expressing a BACH GPCR
polypeptide; or (d) antibody to a BACH GPCR polypeptide. The screening
kit may optionally comprise instructions for use.

Transgenic Animals

[0217]The present invention further encompasses transgenic animals capable
of expressing natural or recombinant BACH GPCR, or a homologue, variant
or derivative, at elevated or reduced levels compared to the normal
expression level. Included are transgenic animals ("BACH knockout"s)
which do not express functional BACH receptor. The BACH knockouts may
arise as a result of functional disruption of the BACH gene or any
portion of that gene, including one or more loss of function mutations,
including a deletion or replacement, of the BACH gene. The mutations
include single point mutations, and may target coding or non-coding
regions of BACH.

[0218]Preferably, such a transgenic animal is a non-human mammal, such as
a pig, a sheep or a rodent. Most preferably the transgenic animal is a
mouse or a rat. Such transgenic animals may be used in screening
procedures to identify agonists and/or antagonists of BACH GPCR, as well
as to test for their efficacy as treatments for diseases in vivo.

[0219]Mice which are null for BACH may be used for various purposes. For
example, transgenic animals that have been engineered to be deficient in
the production of BACH GPCR may be used in assays to identify agonists
and/or antagonists of BACH GPCR. One assay is designed to evaluate a
potential drug (aa candidate ligand or compound) to determine if it
produces a physiological response in the absence of BACH GPCR receptors.
This may be accomplished by administering the drug to a transgenic animal
as discussed above, and then assaying the animal for a particular
response. Although any physiological parameter could be measured in this
assay, preferred responses include one or more of the following: changes
to disease resistance; altered inflammatory responses; altered tumour
susceptability: a change in blood pressure; neovascularization; a change
in eating behavior; a change in body weight; a change in bone density; a
change in body temperature; insulin secretion; gonadotropin secretion;
nasal and bronchial secretion; vasoconstriction; loss of memory; anxiety;
hyporeflexia or hyperreflexia; pain or stress responses.

[0220]Tissues derived from the BACH knockout animals may be used in
receptor binding assays to determine whether the potential drug (a
candidate ligand or compound) binds to the BACH receptor. Such assays can
be conducted by obtaining a first receptor preparation from the
transgenic animal engineered to be deficient in BACH receptor production
and a second receptor preparation from a source known to bind any
identified BACH ligands or compounds. In general, the first and second
receptor preparations will be similar in all respects except for the
source from which they are obtained. For example, if brain tissue from a
transgenic animal (such as described above and below) is used in an
assay, comparable brain tissue from a normal (wild type) animal is used
as the source of the second receptor preparation. Each of the receptor
preparations is incubated with a ligand known to bind to BACH receptors,
both alone and in the presence of the candidate ligand or compound.
Preferably, the candidate ligand or compound will be examined at several
different concentrations.

[0221]The extent to which binding by the known ligand is displaced by the
test compound is determined for both the first and second receptor
preparations. Tissues derived from transgenic animals may be used in
assays directly or the tissues may be processed to isolate membranes or
membrane proteins, which are themselves used in the assays. A preferred
transgenic animal is the mouse. The ligand may be labeled using any means
compatible with binding assays. This would include, without limitation,
radioactive, enzymatic, fluorescent or chemiluminescent labeling (as well
as other labelling techniques as described in further detail above).

[0222]Furthermore, antagonists of BACH GPCR receptor may be identified by
administering candidate compounds, etc, to wild type animals expressing
functional BACH, and animals identified which exhibit any of the
phenotypic characteristics associated with reduced or abolished
expression of BACH receptor function.

[0223]Detailed methods for generating non-human transgenic animal are
described in further detail below. Transgenic gene constructs can be
introduced into the germ line of an animal to make a transgenic mammal.
For example, one or several copies of the construct may be incorporated
into the genome of a mammalian embryo by standard transgenic techniques.

[0224]In an exemplary embodiment, the transgenic non-human animals of the
invention are produced by introducing transgenes into the germline of the
non-human animal. Embryonal target cells at various developmental stages
can be used to introduce transgenes. Different methods are used depending
on the stage of development of the embryonal target cell. The specific
line(s) of any animal used to practice this invention are selected for
general good health, good embryo yields, good pronuclear visibility in
the embryo, and good reproductive fitness. In addition, the haplotype is
a significant factor.

[0225]Introduction of the transgene into the embryo can be accomplished by
any means known in the art such as, for example, microinjection,
electroporation, or lipofection. For example, the BACH receptor transgene
can be introduced into a mammal by microinjection of the construct into
the pronuclei of the fertilized mammalian egg(s) to cause one or more
copies of the construct to be retained in the cells of the developing
mammal(s). Following introduction of the transgene construct into the
fertilized egg, the egg may be incubated in vitro for varying amounts of
time, or reimplanted into the surrogate host, or both. In vitro
incubation to maturity is within the scope of this invention. One common
method in to incubate the embryos in vitro for about 1-7 days, depending
on the species, and then reimplant them into the surrogate host.

[0226]The progeny of the transgenically manipulated embryos can be tested
for the presence of the construct by Southern blot analysis of the
segment of tissue. If one or more copies of the exogenous cloned
construct remains stably integrated into the genome of such transgenic
embryos, it is possible to establish permanent transgenic mammal lines
carrying the transgenically added construct.

[0227]The litters of transgenically altered mammals can be assayed after
birth for the incorporation of the construct into the genome of the
offspring. Preferably, this assay is accomplished by hybridizing a probe
corresponding to the DNA sequence coding for the desired recombinant
protein product or a segment thereof onto chromosomal material from the
progeny. Those mammalian progeny found to contain at least one copy of
the construct in their genome are grown to maturity.

[0228]For the purposes of this invention a zygote is essentially the
formation of a diploid cell which is capable of developing into a
complete organism. Generally, the zygote will be comprised of an egg
containing a nucleus formed, either naturally or artificially, by the
fusion of two haploid nuclei from a gamete or gametes. Thus, the gamete
nuclei must be ones which are naturally compatible, i.e., ones which
result in a viable zygote capable of undergoing differentiation and
developing into a functioning organism. Generally, a euploid zygote is
preferred. If an aneuploid zygote is obtained, then the number of
chromosomes should not vary by more than one with respect to the euploid
number of the organism from which either gamete originated.

[0229]In addition to similar biological considerations, physical ones also
govern the amount (e.g., volume) of exogenous genetic material which can
be added to the nucleus of the zygote or to the genetic material which
forms a part of the zygote nucleus. If no genetic material is removed,
then the amount of exogenous genetic material which can be added is
limited by the amount which will be absorbed without being physically
disruptive. Generally, the volume of exogenous genetic material inserted
will not exceed about 10 picoliters. The physical effects of addition
must not be so great as to physically destroy the viability of the
zygote. The biological limit of the number and variety of DNA sequences
will vary depending upon the particular zygote and functions of the
exogenous genetic material and will be readily apparent to one skilled in
the art, because the genetic material, including the exogenous genetic
material, of the resulting zygote must be biologically capable of
initiating and maintaining the differentiation and development of the
zygote into a functional organism.

[0230]The number of copies of the transgene constructs which are added to
the zygote is dependent upon the total amount of exogenous genetic
material added and will be the amount which enables the genetic
transformation to occur. Theoretically only one copy is required;
however, generally, numerous copies are utilized, for example,
1,000-20,000 copies of the transgene construct, in order to insure that
one copy is functional. As regards the present invention, there will
often be an advantage to having more than one functioning copy of each of
the inserted exogenous DNA sequences to enhance the phenotypic expression
of the exogenous DNA sequences.

[0231]Any technique which allows for the addition of the exogenous genetic
material into nucleic genetic material can be utilized so long as it is
not destructive to the cell, nuclear membrane or other existing cellular
or genetic structures. The exogenous genetic material is preferentially
inserted into the nucleic genetic material by microinjection.
Microinjection of cells and cellular structures is known and is used in
the art.

[0232]Reimplantation is accomplished using standard methods. Usually, the
surrogate host is anesthetized, and the embryos are inserted into the
oviduct. The number of embryos implanted into a particular host will vary
by species, but will usually be comparable to the number of off spring
the species naturally produces.

[0233]Transgenic offspring of the surrogate host may be screened for the
presence and/or expression of the transgene by any suitable method.
Screening is often accomplished by Southern blot or Northern blot
analysis, using a probe that is complementary to at least a portion of
the transgene. Western blot analysis using an antibody against the
protein encoded by the transgene may be employed as an alternative or
additional method for screening for the presence of the transgene
product. Typically, DNA is prepared from tail tissue and analyzed by
Southern analysis or PCR for the transgene. Alternatively, the tissues or
cells believed to express the transgene at the highest levels are tested
for the presence and expression of the transgene using Southern analysis
or PCR, although any tissues or cell types may be used for this analysis.

[0234]Alternative or additional methods for evaluating the presence of the
transgene include, without limitation, suitable biochemical assays such
as enzyme and/or immunological assays, histological stains for particular
marker or enzyme activities, flow cytometric analysis, and the like.
Analysis of the blood may also be useful to detect the presence of the
transgene product in the blood, as well as to evaluate the effect of the
transgene on the levels of various types of blood cells and other blood
constituents.

[0235]Progeny of the transgenic animals may be obtained by mating the
transgenic animal with a suitable partner, or by in vitro fertilization
of eggs and/or sperm obtained from the transgenic animal. Where mating
with a partner is to be performed, the partner may or may not be
transgenic and/or a knockout; where it is transgenic, it may contain the
same or a different transgene, or both. Alternatively, the partner may be
a parental line. Where in vitro fertilization is used, the fertilized
embryo may be implanted into a surrogate host or incubated in vitro, or
both. Using either method, the progeny may be evaluated for the presence
of the transgene using methods described above, or other appropriate
methods.

[0236]The transgenic animals produced in accordance with the present
invention will include exogenous genetic material. As set out above, the
exogenous genetic material will, in certain embodiments, be a DNA
sequence which results in the production of a BACH GPCR receptor.
Further, in such embodiments the sequence will be attached to a
transcriptional control element, e.g., a promoter, which preferably
allows the expression of the transgene product in a specific type of
cell.

[0237]Retroviral infection can also be used to introduce transgene into a
non-human animal. The developing non-human embryo can be cultured in
vitro to the blastocyst stage. During this time, the blastomeres can be
targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
Efficient infection of the blastomeres is obtained by enzymatic treatment
to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The
viral vector system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et al.
(1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS
82:6148-6152). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells (Van
der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
Alternatively, infection can be performed at a later stage. Virus or
virus-producing cells can be injected into the blastocoele (Jahner et al.
(1982) Nature 298:623-628). Most of the founders will be mosaic for the
transgene since incorporation occurs only in a subset of the cells which
formed the transgenic non-human animal. Further, the founder may contain
various retroviral insertions of the transgene at different positions in
the genome which generally will segregate in the offspring. In addition,
it is also possible to introduce transgenes into the germ line by
intrauterine retroviral infection of the midgestation embryo (Jahner et
al. (1982) supra).

[0238]A third type of target cell for transgene introduction is the
embryonal stem cell (ES). ES cells are obtained from pre-implantation
embryos cultured in vitro and fused with embryos (Evans et al. (1981)
Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et
al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature
322:445-448). Transgenes can be efficiently introduced into the ES cells
by DNA transfection or by retrovirus-mediated transduction. Such
transformed ES cells can thereafter be combined with blastocysts from a
non-human animal. The ES cells thereafter colonize the embryo and
contribute to the germ line of the resulting chimeric animal. For review
see Jaenisch, R. (1988) Science 240:1468-1474.

[0239]We also provide non-human transgenic animals, where the transgenic
animal is characterized by having an altered BACH gene, preferably as
described above, as models for BACH receptor function. Alterations to the
gene include deletions or other loss of function mutations, introduction
of an exogenous gene having a nucleotide sequence with targeted or random
mutations, introduction of an exogenous gene from another species, or a
combination thereof. The transgenic animals may be either homozygous or
heterozygous for the alteration. The animals and cells derived therefrom
are useful for screening biologically active agents that may modulate
BACH receptor function. The screening methods are of particular use for
determining the specificity and action of potential therapies for BACH
associated diseases including trigeminal neuralgia, orofacial pain, pain
associated with toothache, irritable bowel syndrome, Barrett's
oesophagus, glaucoma, pain associated with cancer, diabetic neuropathies,
Herpes infections, HIV infections, migraine and skin sensitivity
associated with migraine, allodynia, toothache, neuroma (whether caused
by amputation, nerve transaction or trauma), nerve compression (caused by
tumours, entrapment or crush), pain due to damage of the spinal cord or
brain; dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign
essential tremor, chorea, epilepsy or ballismus, for example occurring
through stroke, trauma, degeneration or malignancy; dry-eye disorders,
cystic fibrosis, hyperactive bladder, hypercholesterolaemia,
dislipdaemias and obesity. The animals are useful as a model to
investigate the role of BACH receptors in normal brain, heart, spleen and
liver function.

[0240]Another aspect of the invention pertains to a transgenic nonhuman
animal having a functionally disrupted endogenous BACH gene but which
also carries in its genome, and expresses, a transgene encoding a
heterologous BACH protein (i.e., a BACH from another species).
Preferably, the animal is a mouse and the heterologous BACH is a human
BACH. An animal, or cell lines derived from such an animal of the
invention, which has been reconstituted with human BACH, can be used to
identify agents that inhibit human BACH in vivo and in vitro. For
example, a stimulus that induces signalling through human BACH can be
administered to the animal, or cell line, in the presence and absence of
an agent to be tested and the response in the animal, or cell line, can
be measured. An agent that inhibits human BACH in vivo or in vitro can be
identified based upon a decreased response in the presence of the agent
compared to the response in the absence of the agent.

[0241]The present invention also provides for a BACH GPCR deficient
transgenic non-human animal (a "BACH GPCR knock-out" or a "BACH null").
Such an animal is one which expresses lowered or no BACH GPCR activity,
preferably as a result of an endogenous BACH GPCR genomic sequence being
disrupted or deleted. The endogenous BACH GPCR genomic sequence may be
replaced by a null allele, which may comprise non-functional portions of
the wild-type BACH sequence. For example, the endogenous BACH GPCR
genomic sequence may be replaced by an allele of BACH comprising a
disrupting sequence which may comprise heterologous sequences, for
example, reporter sequences and/or selectable markers. Preferably, the
endogenous BACH GPCR genomic sequence in a BACH knock-out mouse is
replaced by an allele of BACH in which one or more, preferably all, of
the transmembrane sequences is replaced by such a disrupting sequence,
preferably a lacZ sequence and a neomycin resistance sequence.
Preferably, the genomic BACH sequence which is functionally disrupted
comprises a mouse BACH genomic sequence shown in SEQ ID NO: 10.

[0242]Preferably, such an animal expresses no GPCR activity. More
preferably, the animal expresses no activity of the BACH GPCR shown as
SEQ ID NO: 3 or SEQ ID NO: 5. BACH GPCR knock-outs may be generated by
various means known in the art, as described in further detail below. A
specific description of the construction of a BACH knock-out mouse is
disclosed in Example 2 below.

[0243]The present invention also pertains to a nucleic acid construct for
functionally disrupting a BACH gene in a host cell. The nucleic acid
construct comprises: a) a non-homologous replacement portion; b) a first
homology region located upstream of the non-homologous replacement
portion, the first homology region having a nucleotide sequence with
substantial identity to a first BACH gene sequence; and c) a second
homology region located downstream of the non-homologous replacement
portion, the second homology region having a nucleotide sequence with
substantial identity to a second BACH gene sequence, the second BACH gene
sequence having a location downstream of the first BACH gene sequence in
a naturally occurring endogenous BACH gene. Additionally, the first and
second homology regions are of sufficient length for homologous
recombination between the nucleic acid construct and an endogenous BACH
gene in a host cell when the nucleic acid molecule is introduced into the
host cell. In a preferred embodiment, the non-homologous replacement
portion comprises an expression reporter, preferably including lacZ and a
positive selection expression cassette, preferably including a neomycin
phosphotransferase gene operatively linked to a regulatory element(s).

[0244]Preferably, the first and second BACH gene sequences are derived
from SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4, or a mouse BACH genomic
sequence (SEQ ID NO: 10), or a homologue, variant or derivative thereof.
preferably, the construct comprises a structure depicted in FIG. 4.

[0245]Another aspect of the invention pertains to recombinant vectors into
which the nucleic acid construct of the invention has been incorporated.
Yet another aspect of the invention pertains to host cells into which the
nucleic acid construct of the invention has been introduced to thereby
allow homologous recombination between the nucleic acid construct and an
endogenous BACH gene of the host cell, resulting in functional disruption
of the endogenous BACH gene. The host cell can be a mammalian cell that
normally expresses BACH from the liver, brain, spleen or heart, or a
pluripotent cell, such as a mouse embryonic stem cell. Further
development of an embryonic stem cell into which the nucleic acid
construct has been introduced and homologously recombined with the
endogenous BACH gene produces a transgenic nonhuman animal having cells
that are descendant from the embryonic stem cell and thus carry the BACH
gene disruption in their genome. Animals that carry the BACH gene
disruption in their germline can then be selected and bred to produce
animals having the BACH gene disruption in all somatic and germ cells.
Such mice can then be bred to homozygosity for the BACH gene disruption.

[0248]Murine BACH genomic clones are isolated from a mouse large insert
PAC library obtained from HGMP (Hinxton, UK) using the human open reading
frame cDNA sequence (SEQ ID NO: 1) as a probe using standard techniques.
The isolated murine BACH genomic clones are then restriction mapped in
the region of the BACH gene using small oligonucleotide probes and
standard techniques. A mouse genomic BACH sequence is depicted as SEQ ID
NO: 10.

[0249]The murine genomic locus is partially sequenced to enable the design
of homologous arms to clone into the targeting vector. The murine BACH
gene is a single exon gene. A 5' homologous arm and a 3' homologous arm
are amplified by PCR and the fragment cloned into the targeting vector.
Any suitable size may be chosen for the length of these arms to enable
homologous recombination; for example, the 5' arm may be between 1 kb and
to 2 kb, for example 1.15 kb, while the 3' arm may be about 4 kb in size.

[0250]The position of these arms is chosen to functionally disrupt the
BACH gene by deleting the seven transmembrane spanning regions. A
targeting vector is prepared where the deleted BACH sequence is replaced
with non-homologous sequences composed of an endogenous gene expression
reporter (an in frame fusion with lacZ) upstream of a selection cassette
composed of a self promoted neomycin phosphotransferase (neo) gene in the
same orientation as the BACH gene.

[0251]Transfection and Analysis of Embryonal Stem Cells

[0252]Embryonal stem cells (Evans and Kaufman, 1981) are cultured on a
neomycin resistant embryonal fibroblast feeder layer grown in Dulbecco's
Modified Eagles medium supplemented with 20% Fetal Calf Serum, 10%
new-born calf serum, 2 mM glutamine, non-essential amino acids, 100 μM
2-mercaptoethanol and 500 u/ml leukemia inhibitory factor. Medium is
changed daily and ES cells are subcultured every three days.
5×106 ES cells are transfected with 5 μg of linearized
plasmid by electroporation (25 μF capacitance and 400 Volts). 24 hours
following electroporation the transfected cells are cultured for 9 days
in medium containing 200 μg/ml neomycin. Clones are picked into 96
well plates, replicated and expanded before being screened by PCR to
identify clones in which homologous recombination had occurred between
the endogenous BACH gene and the targeting construct. From 200 picked
clones 7 targets are identified. These clones where expanded to allow
replicas to be frozen and sufficient high quality DNA to be prepared for
Southern blot confirmation of the targeting event using external 5' and
3' probes, all using standard procedures (Russ et al, 2000)

Generation of BACH GPCR Deficient Mice

[0253]C57BL/6 female and male mice are mated and blastocysts are isolated
at 3.5 days of gestation. 10-12 cells from a chosen clone are injected
per blastocyst and 7-8 blastocysts are implanted in the uterus of a
pseudopregnant F1 female. Five chimeric pups are born of which one male
is 100% agouti (indicating cells descendent from the targeted clone).
This male chimera is mated with female and MF1 and 129 mice, and germline
transmission is determined by the agouti coat color and by PCR genotyping
respectively.

Antibodies

[0254]The present invention further provides for antibodies which bind to
a BACH polypeptide, fragment, homologue, variant or derivative thereof.
Particularly BACH expression, and in particular in diagnosing a BACH GPCR
associated disease. Other preferred antibodies include those which have
therapeutic activity, i.e., which are may be used in a therapeutic manner
to treat, manage or prevent any BACH GPCR associated disease.

[0255]For the purposes of this invention, the term "antibody", unless
specified to the contrary, includes but is not limited to, polyclonal,
monoclonal, chimeric, single chain, Fab fragments and fragments produced
by a Fab expression library. Such fragments include fragments of whole
antibodies which retain their binding activity for a target substance,
Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies
(scFv), fusion proteins and other synthetic proteins which comprise the
antigen-binding site of the antibody. The antibodies and fragments
thereof may be humanised antibodies, for example as described in
EP-A-239400. Furthermore, antibodies with fully human variable regions
(or their fragments), for example, as described in U.S. Pat. Nos.
5,545,807 and 6,075,181 may also be used. Neutralizing antibodies, i.e.,
those which inhibit any biological activity of BACH, are especially
preferred for diagnostics and therapeutics.

[0256]Antibodies may be produced by standard techniques, such as by
immunisation or by using a phage display library.

[0257]A polypeptide or peptide of the present invention may be used to
develop an antibody by known techniques. Such an antibody may be capable
of binding specifically to the BACH GPCR protein or homologue, fragment,
etc.

[0258]If polyclonal antibodies are desired, a selected mammal (e.g.,
mouse, rabbit, goat, horse, etc.) may be immunised with an immunogenic
composition comprising a polypeptide or peptide of the present invention.
Depending on the host species, various adjuvants may be used to increase
immunological response. Such adjuvants include, but are not limited to,
Freund's, mineral gels such as aluminium hydroxide, and surface active
substances such as lysolecithin, pluronic polyols, polyanions, peptides,
oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacilli
Calmette-Guerin) and Corynebacterium parvum are potentially useful human
adjuvants which may be employed if purified the substance amino acid
sequence is administered to immunologically compromised individuals for
the purpose of stimulating systemic defense.

[0259]Serum from the immunised animal is collected and treated according
to known procedures. If serum containing polyclonal antibodies to an
epitope obtainable from a polypeptide of the present invention contains
antibodies to other antigens, the polyclonal antibodies can be purified
by immunoaffinity chromatography. Techniques for producing and processing
polyclonal antisera are known in the art. In order that such antibodies
may be made, the invention also provides amino acid sequences of the
invention or fragments thereof haptenised to another amino acid sequence
for use as immunogens in animals or humans.

[0260]Monoclonal antibodies directed against epitopes obtainable from a
polypeptide or peptide of the present invention can also be readily
produced by one skilled in the art. The general methodology for making
monoclonal antibodies by hybridomas is well known Immortal
antibody-producing cell lines can be created by cell fusion, and also by
other techniques such as direct transformation of B lymphocytes with
oncogenic DNA, or transfection with Epstein-Ban virus. Panels of
monoclonal antibodies produced against orbit epitopes can be screened for
various properties; i.e., for isotype and epitope affinity.

[0262]In addition, techniques developed for the production of "chimeric
antibodies", the splicing of mouse antibody genes to human antibody genes
to obtain a molecule with appropriate antigen specificity and biological
activity can be used (Morrison et al (1984) Proc Natl Acad Sci
81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al
(1985) Nature 314:452-454). Alternatively, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,779) can be
adapted to produce the substance specific single chain antibodies.

[0263]Antibodies, both monoclonal and polyclonal, which are directed
against epitopes obtainable from a polypeptide or peptide of the present
invention are particularly useful in diagnosis, and those which are
neutralising are useful in passive immunotherapy. Monoclonal antibodies,
in particular, may be used to raise anti-idiotype antibodies.
Anti-idiotype antibodies are immunoglobulins which carry an "internal
image" of the substance and/or agent against which protection is desired.
Techniques for raising anti-idiotype antibodies are known in the art.
These anti-idiotype antibodies may also be useful in therapy.

[0264]Antibodies may also be produced by inducing in vivo production in
the lymphocyte population or by screening recombinant immunoglobulin
libraries or panels of highly specific binding reagents as disclosed in
Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and
Milstein C (1991; Nature 349:293-299).

[0265]Antibody fragments which contain specific binding sites for the
polypeptide or peptide may also be generated. For example, such fragments
include, but are not limited to, the F(ab')2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal Fab
fragments with the desired specificity (Huse W D et al (1989) Science
256:1275-128 1).

[0266]Techniques for the production of single chain antibodies (U.S. Pat.
No. 4,946,778) can also be adapted to produce single chain antibodies to
polypeptides of this invention. Also, transgenic mice, or other organisms
including other mammals, may be used to express humanized antibodies.

[0267]The above-described antibodies may be employed to isolate or to
identify clones expressing the polypeptide or to purify the polypeptides
by affinity chromatography.

[0269]This invention also relates to the use of BACH GPCR polynucleotides
and polypeptides (as well as homologues, variants and derivatives
thereof) for use in diagnosis as diagnostic reagents or in genetic
analysis. Nucleic acids complementary to or capable of hybridising to
BACH GPCR nucleic acids (including homologues, variants and derivatives),
as well as antibodies against BACH polypeptides are also useful in such
assays.

[0270]We provide for a natural variant of BACH polypeptide or nucleic
acid, and the use of such a natural variant in diagnosis of BACH
associated disease. BACH polymorphisms may include differences at the
nucleic acid level, which may or may not reflect differences in the amino
acid level. Preferably, such BACH variants or mutants are such that they
include changes in the amino acid level. However, the invention also
encompasses BACH polymorphisms which occur in non-coding regions, for
example, expression control regions such as promoters and enhancers.

[0271]Polymorphisms in BACH include deletions of one or more nucleic
acids, insertions of one or more nucleic acids, inversions, etc.
Preferably, BACH polymorphisms comprise single nucleotide polymorphisms.

[0272]Polymorphisms in BACH may be identified by comparing sequences at
the appropriate level (whether nucleic acid or protein) between
individuals in a population. Differences in sequences may be reflected in
different physical properties, and techniques for detecting these may
rely on detection of changes in physical properties. For example, single
nucleotide polymorphisms may be detected as restriction fragment length
polymorphisms (i.e., difference in susceptibility to digestion by a
restriction enzyme). Furthermore, SNPS may affect the migration or
mobility of a nucleic acid fragment or protein fragment in a gel.

[0273]Non-coding polymorphisms in BACH may be identified by sequencing
non-coding regions of BACH. For example, control regions of the BACH
gene, such as enhancers and promoters may be sequenced to identify
polymorphisms. The effect of such non-coding polymorphisms on the
expression level of BACH may be determined by constructing transgenic
mice (as described below) comprising the mutant BACH sequences, or by
generating expression constructs and transfection into cell lines. In
each case, the expression level of BACH is detected, by RT-PCR or
antibody Western staining, to determine the effect of the mutation in the
control of expression of BACH. Useful BACH polymorphisms are those which
modulate the level of expression, whether by up-regulation or
down-regulation of BACH levels.

[0274]Accordingly, this invention provides for a variant or mutant or
polymorphism in a non-coding region of BACH, preferably in a control
region of BACH, preferably in a promoter and/or enhancer of BACH, which
is capable of modulating the level of expression of BACH in an organism.
The invention also provides for a set of two or more of such mutants or
variants or polymorphisms, preferably non-coding polymorphisms. The
invention also provides for the use of such variants or polymorphisms or
sets of variants to identify nucleic acid and/or amino acid positions, in
which changes to such positions affect the level of expression of BACH.
The invention also provides for a transgenic animal comprising a variant
or mutant or polymorphism of BACH, preferably, a non-coding polymorphism.

[0275]Detection of a mutated form of the BACH GPCR gene associated with a
dysfunction will provide a diagnostic tool that can add to or define a
diagnosis of a disease or susceptibility to a disease which results from
under-expression, over-expression or altered expression of BACH GPCR.
Individuals carrying mutations in the BACH GPCR gene (including control
sequences) may be detected at the DNA level by a variety of techniques.

[0276]For example, DNA may be isolated from a patient and the DNA
polymorphism pattern of BACH determined. The identified pattern is
compared to controls of patients known to be suffering from a disease
associated with over-, under- or abnormal expression of BACH. Patients
expressing a genetic polymorphism pattern associated with BACH associated
disease may then be identified. Genetic analysis of the BACH GPCR gene
may be conducted by any technique known in the art. For example,
individuals may be screened by determining DNA sequence of a BACH allele,
by RFLP or SNP analysis, etc. Patients may be identified as having a
genetic predisposition for a disease associated with the over-, under-,
or abnormal expression of BACH by detecting the presence of a DNA
polymorphism in the gene sequence for BACH or any sequence controlling
its expression.

[0277]Patients so identified can then be treated to prevent the occurrence
of BACH associated disease, or more aggressively in the early stages of
BACH associated disease to prevent the further occurrence or development
of the disease. BACH associated diseases include any one of trigeminal
neuralgia, orofacial pain, pain associated with toothache, irritable
bowel syndrome, Barrett's oesophagus, glaucoma, pain associated with
cancer, diabetic neuropathies, Herpes infections, HIV infections,
migraine and skin sensitivity associated with migraine, allodynia,
toothache, neuroma (whether caused by amputation, nerve transaction or
trauma), nerve compression (caused by tumours, entrapment or crush), pain
due to damage of the spinal cord or brain; dementia, dyslexia,
dyskinesias, tremor, Parkinson's, benign essential tremor, chorea,
epilepsy and ballismus, for example occurring through stroke, trauma,
degeneration or malignancy; cystic fibrosis and hyperactive bladder.

[0278]The present invention further discloses a kit for the identification
of a patient's genetic polymorphism pattern associated with BACH
associated disease. The kit includes DNA sample collecting means and
means for determining a genetic polymorphism pattern, which is then
compared to control samples to determine a patient's susceptibility to
BACH associated disease. Kits for diagnosis of a BACH associated disease
comprising BACH polypeptide and/or an antibody against such a polypeptide
(or fragment of it) are also provided.

[0279]Nucleic acids for diagnosis may be obtained from a subject's cells,
such as from blood, urine, saliva, tissue biopsy or autopsy material. In
a preferred embodiment, the DNA is obtained from blood cells obtained
from a finger prick of the patient with the blood collected on absorbent
paper. In a further preferred embodiment, the blood is collected on an
AmpliCard® (University of Sheffield, Department of Medicine and
Pharmacology, Royal Hallamshire Hospital, Sheffield, England S10 2JF).

[0280]The DNA may be used directly for detection or may be amplified
enzymatically by using PCR or other amplification techniques prior to
analysis. Oligonucleotide DNA primers that target the specific
polymorphic DNA region within the genes of interest may be prepared so
that in the PCR reaction amplification of the target sequences is
achieved. RNA or cDNA may also be used as templates in similar fashion.
The amplified DNA sequences from the template DNA may then be analyzed
using restriction enzymes to determine the genetic polymorphisms present
in the amplified sequences and thereby provide a genetic polymorphism
profile of the patient. Restriction fragments lengths may be identified
by gel analysis. Alternatively, or in conjunction, techniques such as SNP
(single nucleotide polymorphisms) analysis may be employed.

[0281]Deletions and insertions can be detected by a change in size of the
amplified product in comparison to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to labeled BACH GPCR
nucleotide sequences. Perfectly matched sequences can be distinguished
from mismatched duplexes by RNase digestion or by differences in melting
temperatures. DNA sequence differences may also be detected by
alterations in electrophoretic mobility of DNA fragments in gels, with or
without denaturing agents, or by direct DNA sequencing. See, eg., Myers
et al, Science (1985)230:1242. Sequence changes at specific locations may
also be revealed by nuclease protection assays, such as RNase and
S1protection or the chemical cleavage method. See Cotton et al., Proc
Natl Acad Sci USA (1985) 85: 4397-4401. In another embodiment, an array
of oligonucleotides probes comprising the BACH GPCR nucleotide sequence
or fragments thereof can be constructed to conduct efficient screening of
e.g., genetic mutations. Array technology methods are well known and have
general applicability and can be used to address a variety of questions
in molecular genetics including gene expression, genetic linkage, and
genetic variability. (See for example: M. Chee et al., Science, Vol 274,
pp 610-613 (1996)).

[0282]Single strand conformation polymorphism (SSCP) may be used to detect
differences in electrophoretic mobility between mutant and wild type
nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see
also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal
Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control
BACH nucleic acids may be denatured and allowed to renature. The
secondary structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility enables
the detection of even a single base change. The DNA fragments may be
labeled or detected with labeled probes. The sensitivity of the assay may
be enhanced by using RNA (rather than DNA), in which the secondary
structure is more sensitive to a change in sequence. In a preferred
embodiment, the subject method utilizes heteroduplex analysis to separate
double stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0284]The presence of BACH GPCR polypeptides and nucleic acids may be
detected in a sample. Thus, infections and diseases as listed above can
be diagnosed by methods comprising determining from a sample derived from
a subject an abnormally decreased or increased level of the BACH GPCR
polypeptide or BACH GPCR mRNA. The sample may comprise a cell or tissue
sample from an organism suffering or suspected to be suffering from a
disease associated with increased, reduced or otherwise abnormal BACH
GPCR expression, including spatial or temporal changes in level or
pattern of expression. The level or pattern of expression of BACH in an
organism suffering from or suspected to be suffering from such a disease
may be usefully compared with the level or pattern of expression in a
normal organism as a means of diagnosis of disease.

[0285]In general therefore, the invention includes a method of detecting
the presence of a nucleic acid comprising a BACH GPCR nucleic acid in a
sample, by contacting the sample with at least one nucleic acid probe
which is specific for said nucleic acid and monitoring said sample for
the presence of the nucleic acid. For example, the nucleic acid probe may
specifically bind to the BACH GPCR nucleic acid, or a portion of it, and
binding between the two detected; the presence of the complex itself may
also be detected. Furthermore, the invention encompasses a method of
detecting the presence of a BACH GPCR polypeptide by contacting a cell
sample with an antibody capable of binding the polypeptide and monitoring
said sample for the presence of the polypeptide. This may conveniently be
achieved by monitoring the presence of a complex formed between the
antibody and the polypeptide, or monitoring the binding between the
polypeptide and the antibody. Methods of detecting binding between two
entities are known in the art, and include FRET (fluorescence resonance
energy transfer), surface plasmon resonance, etc.

[0286]Decreased or increased expression can be measured at the RNA level
using any of the methods well known in the art for the quantitation of
polynucleotides, such as, for example, PCR, RT-PCR, RNase protection,
Northern blotting and other hybridization methods. Assay techniques that
can be used to determine levels of a protein, such as a BACH GPCR, in a
sample derived from a host are well-known to those of skill in the art.
Such assay methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis and ELISA assays.

[0288]The diagnostic kit comprises a BACH GPCR polynucleotide or a
fragment thereof; a complementary nucleotide sequence; a BACH GPCR
polypeptide or a fragment thereof, or an antibody to a BACH GPCR
polypeptide.

Chromosome Assays

[0289]The nucleotide sequences of the present invention are also valuable
for chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. As described above, human BACH GPCR is found to map to Homo
sapiens chromosome 12p13.3.

[0290]The mapping of relevant sequences to chromosomes according to the
present invention is an important first step in correlating those
sequences with gene associated disease. Once a sequence has been mapped
to a precise chromosomal location, the physical position of the sequence
on the chromosome can be correlated with genetic map data. Such data are
found, for example, in V. McKusick, Mendelian heritance in Man (available
on line through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to the same
chromosomal region are then identified through linkage analysis
(coinheritance of physically adjacent genes).

[0291]The differences in the cDNA or genomic sequence between affected and
unaffected individuals can also be determined If a mutation is observed
in some or all of the affected individuals but not in any normal
individuals, then the mutation is likely to be the causative agent of the
disease.

Prophylactic and Therapeutic Methods

[0292]This invention provides methods of treating an abnormal conditions
related to both an excess of and insufficient amounts of BACH GPCR
activity.

[0293]If the activity of BACH GPCR is in excess, several approaches are
available. One approach comprises administering to a subject an inhibitor
compound (antagonist) as hereinabove described along with a
pharmaceutically acceptable carrier in an amount effective to inhibit
activation by blocking binding of ligands to the BACH GPCR, or by
inhibiting a second signal, and thereby alleviating the abnormal
condition.

[0294]In another approach, soluble forms of BACH GPCR polypeptides still
capable of binding the ligand in competition with endogenous BACH GPCR
may be administered. Typical embodiments of such competitors comprise
fragments of the BACH GPCR polypeptide.

[0295]In still another approach, expression of the gene encoding
endogenous BACH GPCR can be inhibited using expression blocking
techniques. Known such techniques involve the use of antisense sequences,
either internally generated or separately administered. See, for example,
O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).
Alternatively, oligonucleotides which form triple helices with the gene
can be supplied. See, for example, Lee et al., Nucleic Acids Res (1979)
6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Science
(1991) 251:1360. These oligomers can be administered per se or the
relevant oligomers can be expressed in vivo.

[0296]For treating abnormal conditions related to an under-expression of
BACH GPCR and its activity, several approaches are also available. One
approach comprises administering to a subject a therapeutically effective
amount of a compound which activates BACH GPCR, i.e., an agonist as
described above, in combination with a pharmaceutically acceptable
carrier, to thereby alleviate the abnormal condition. Alternatively, gene
therapy may be employed to effect the endogenous production of BACH GPCR
by the relevant cells in the subject. For example, a polynucleotide of
the invention may be engineered for expression in a replication defective
retroviral vector, as discussed above. The retroviral expression
construct may then be isolated and introduced into a packaging cell
transduced with a retroviral plasmid vector containing RNA encoding a
polypeptide of the present invention such that the packaging cell now
produces infectious viral particles containing the gene of interest.
These producer cells may be administered to a subject for engineering
cells in vivo and expression of the polypeptide in vivo. For overview of
gene therapy, see Chapter 20, Gene Therapy and other Molecular
Genetic-based Therapeutic Approaches, (and references cited therein) in
Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific
Publishers Ltd (1996).

Formulation and Administration

[0297]Peptides, such as the soluble form of BACH GPCR polypeptides, and
agonists and antagonist peptides or small molecules, may be formulated in
combination with a suitable pharmaceutical carrier. Such formulations
comprise a therapeutically effective amount of the polypeptide or
compound, and a pharmaceutically acceptable carrier or excipient. Such
carriers include but are not limited to, saline, buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof. Formulation
should suit the mode of administration, and is well within the skill of
the art. The invention further relates to pharmaceutical packs and kits
comprising one or more containers filled with one or more of the
ingredients of the aforementioned compositions of the invention.

[0298]Polypeptides and other compounds of the present invention may be
employed alone or in conjunction with other compounds, such as
therapeutic compounds.

[0299]Preferred forms of systemic administration of the pharmaceutical
compositions include injection, typically by intravenous injection. Other
injection routes, such as subcutaneous, intramuscular, or
intraperitoneal, can be used. Alternative means for systemic
administration include transmucosal and transdermal administration using
penetrants such as bile salts or fusidic acids or other detergents. In
addition, if properly formulated in enteric or encapsulated formulations,
oral administration may also be possible. Administration of these
compounds may also be topical and/or localize, in the form of salves,
pastes, gels and the like.

[0300]The dosage range required depends on the choice of peptide, the
route of administration, the nature of the formulation, the nature of the
subject's condition, and the judgment of the attending practitioner.
Suitable dosages, however, are in the range of 0.1-100 μg/kg of
subject. Wide variations in the needed dosage, however, are to be
expected in view of the variety of compounds available and the differing
efficiencies of various routes of administration. For example, oral
administration would be expected to require higher dosages than
administration by intravenous injection. Variations in these dosage
levels can be adjusted using standard empirical routines for
optimization, as is well understood in the art.

[0301]Polypeptides used in treatment can also be generated endogenously in
the subject, in treatment modalities often referred to as "gene therapy"
as described above. Thus, for example, cells from a subject may be
engineered with a polynucleotide, such as a DNA or RNA, to encode a
polypeptide ex vivo, and for example, by the use of a retroviral plasmid
vector. The cells are then introduced into the subject.

[0303]The pharmaceutical compositions may be for human or animal usage in
human and veterinary medicine and will typically comprise any one or more
of a pharmaceutically acceptable diluent, carrier, or excipient.
Acceptable carriers or diluents for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
The choice of pharmaceutical carrier, excipient or diluent can be
selected with regard to the intended route of administration and standard
pharmaceutical practice. The pharmaceutical compositions may comprise
as--or in addition to--the carrier, excipient or diluent any suitable
binder(s), lubricant(s), suspending agent(s), coating agent(s),
solubilising agent(s).

[0304]Preservatives, stabilizers, dyes and even flavoring agents may be
provided in the pharmaceutical composition. Examples of preservatives
include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
Antioxidants and suspending agents may be also used.

[0305]There may be different composition/formulation requirements
dependent on the different delivery systems. By way of example, the
pharmaceutical composition of the present invention may be formulated to
be delivered using a mini-pump or by a mucosal route, for example, as a
nasal spray or aerosol for inhalation or ingestable solution, or
parenterally in which the composition is formulated by an injectable
form, for delivery, by, for example, an intravenous, intramuscular or
subcutaneous route. Alternatively, the formulation may be designed to be
delivered by both routes.

[0306]Where the agent is to be delivered mucosally through the
gastrointestinal mucosa, it should be able to remain stable during
transit though the gastrointestinal tract; for example, it should be
resistant to proteolytic degradation, stable at acid pH and resistant to
the detergent effects of bile.

[0307]Where appropriate, the pharmaceutical compositions can be
administered by inhalation, in the form of a suppository or pessary,
topically in the form of a lotion, solution, cream, ointment or dusting
powder, by use of a skin patch, orally in the form of tablets containing
excipients such as starch or lactose, or in capsules or ovules either
alone or in admixture with excipients, or in the form of elixirs,
solutions or suspensions containing flavouring or colouring agents, or
they can be injected parenterally, for example intravenously,
intramuscularly or subcutaneously. For parenteral administration, the
compositions may be best used in the form of a sterile aqueous solution
which may contain other substances, for example enough salts or
monosaccharides to make the solution isotonic with blood. For buccal or
sublingual administration the compositions may be administered in the
form of tablets or lozenges which can be formulated in a conventional
manner.

[0309]Yet another embodiment of the invention relates to a method of
inducing immunological response in a mammal which comprises delivering a
BACH GPCR polypeptide via a vector directing expression of a BACH GPCR
polynucleotide in vivo in order to induce such an immunological response
to produce antibody to protect said animal from diseases.

[0310]A further embodiment of the invention relates to an
immunological/vaccine formulation (composition) which, when introduced
into a mammalian host, induces an immunological response in that mammal
to a BACH GPCR polypeptide wherein the composition comprises a BACH GPCR
polypeptide or BACH GPCR gene. The vaccine formulation may further
comprise a suitable carrier.

[0311]Since the BACH GPCR polypeptide may be broken down in the stomach,
it is preferably administered parenterally (including subcutaneous,
intramuscular, intravenous, intradermal etc. injection). Formulations
suitable for parenteral administration include aqueous and non-aqueous
sterile injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation instonic with the
blood of the recipient; and aqueous and non-aqueous sterile suspensions
which may include suspending agents or thickening agents. The
formulations may be presented in unit-dose or multi-dose containers, for
example, sealed ampoules and vials and may be stored in a freeze-dried
condition requiring only the addition of the sterile liquid carrier
immediately prior to use. The vaccine formulation may also include
adjuvant systems for enhancing the immunogenicity of the formulation,
such as oil-in water systems and other systems known in the art. The
dosage will depend on the specific activity of the vaccine and can be
readily determined by routine experimentation.

[0312]Vaccines may be prepared from one or more polypeptides or peptides
of the present invention.

[0313]The preparation of vaccines which contain an immunogenic
polypeptide(s) or peptide(s) as active ingredient(s), is known to one
skilled in the art. Typically, such vaccines are prepared as injectables,
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also be
prepared. The preparation may also be emulsified, or the protein
encapsulated in liposomes. The active immunogenic ingredients are often
mixed with excipients which are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol, or the like and
combinations thereof.

[0314]In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH buffering
agents, and/or adjuvants which enhance the effectiveness of the vaccine.
Examples of adjuvants which may be effective include but are not limited
to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine
(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,
referred to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as
MTP-PE), and RIBI, which contains three components extracted from
bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall
skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

[0316]Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel
(aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used.
Only aluminum hydroxide is approved for human use.

[0317]The proportion of immunogen and adjuvant can be varied over a broad
range so long as both are present in effective amounts. For example,
aluminum hydroxide can be present in an amount of about 0.5% of the
vaccine mixture (Al2O3 basis). Conveniently, the vaccines are
formulated to contain a final concentration of immunogen in the range of
from 0.2 to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15
μg/ml.

[0318]After formulation, the vaccine may be incorporated into a sterile
container which is then sealed and stored at a low temperature, for
example 4° C., or it may be freeze-dried. Lyophilisation permits
long-term storage in a stabilised form.

[0319]The vaccines are conventionally administered parenterally, by
injection, for example, either subcutaneously or intramuscularly.
Additional formulations which are suitable for other modes of
administration include suppositories and, in some cases, oral
formulations. For suppositories, traditional binders and carriers may
include, for example, polyalkylene glycols or triglycerides; such
suppositories may be formed from mixtures containing the active
ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral
formulations include such normally employed excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate, and the like. These
compositions take the form of solutions, suspensions, tablets, pills,
capsules, sustained release formulations or powders and contain 10% to
95% of active ingredient, preferably 25% to 70%. Where the vaccine
composition is lyophilised, the lyophilised material may be reconstituted
prior to administration, e.g. as a suspension. Reconstitution is
preferably effected in buffer

[0320]Capsules, tablets and pills for oral administration to a patient may
be provided with an enteric coating comprising, for example, Eudragit
"S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or
hydroxypropylmethyl cellulose.

[0321]The polypeptides of the invention may be formulated into the vaccine
as neutral or salt forms. Pharmaceutically acceptable salts include the
acid addition salts (formed with free amino groups of the peptide) and
which are formed with inorganic acids such as, for example, hydrochloric
or phosphoric acids, or such organic acids such as acetic, oxalic,
tartaric and maleic. Salts formed with the free carboxyl groups may also
be derived from inorganic bases such as, for example, sodium, potassium,
ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and
procaine.

Administration

[0322]Typically, a physician will determine the actual dosage which will
be most suitable for an individual subject and it will vary with the age,
weight and response of the particular patient. The dosages below are
exemplary of the average case. There can, of course, be individual
instances where higher or lower dosage ranges are merited.

[0323]The pharmaceutical and vaccine compositions of the present invention
may be administered by direct injection. The composition may be
formulated for parenteral, mucosal, intramuscular, intravenous,
subcutaneous, intraocular or transdermal administration. Typically, each
protein may be administered at a dose of from 0.01 to 30 mg/kg body
weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1
mg/kg body weight.

[0325]The term "administered" includes but is not limited to delivery by a
mucosal route, for example, as a nasal spray or aerosol for inhalation or
as an ingestable solution; a parenteral route where delivery is by an
injectable form, such as, for example, an intravenous, intramuscular or
subcutaneous route.

[0326]The term "co-administered" means that the site and time of
administration of each of for example, the polypeptide of the present
invention and an additional entity such as adjuvant are such that the
necessary modulation of the immune system is achieved. Thus, whilst the
polypeptide and the adjuvant may be administered at the same moment in
time and at the same site, there may be advantages in administering the
polypeptide at a different time and to a different site from the
adjuvant. The polypeptide and adjuvant may even be delivered in the same
delivery vehicle--and the polypeptide and the antigen may be coupled
and/or uncoupled and/or genetically coupled and/or uncoupled.

[0327]The polypeptide, polynucleotide, peptide, nucleotide, antibody of
the invention and optionally an adjuvant may be administered separately
or co-administered to the host subject as a single dose or in multiple
doses.

[0328]The vaccine composition and pharmaceutical compositions of the
present invention may be administered by a number of different routes
such as injection (which includes parenteral, subcutaneous and
intramuscular injection) intranasal, mucosal, oral, intra-vaginal,
urethral or ocular administration.

[0329]The vaccines and pharmaceutical compositions of the present
invention may be conventionally administered parenterally, by injection,
for example, either subcutaneously or intramuscularly. Additional
formulations which are suitable for other modes of administration include
suppositories and, in some cases, oral formulations. For suppositories,
traditional binders and carriers may include, for example, polyalkylene
glycols or triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%, may be 1%
to 2%. Oral formulations include such normally employed excipients as,
for example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,
and the like. These compositions take the form of solutions, suspensions,
tablets, pills, capsules, sustained release formulations or powders and
contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the
vaccine composition is lyophilised, the lyophilised material may be
reconstituted prior to administration, e.g. as a suspension.
Reconstitution is preferably effected in buffer.

Examples

Example 1

Transgenic BACH Knock-Out Mouse

Construction of BACH Gene Targeting Vector

[0330]Murine BACH genomic clones are isolated from a mouse large insert
PAC library obtained from HGMP (Hinxton, UK) using the human open reading
frame cDNA sequence (SEQ ID NO: 1) as a probe using standard techniques.

[0332]The isolated murine BACH genomic clones are then restriction mapped
in the region of the BACH gene using small oligonucleotide probes and
standard techniques. The murine genomic locus is partially sequenced to
enable the design of homologous arms to clone into the targeting
vector1. The murine BACH gene is a single exon gene. A 1.15 kb 5'
homologous arm and an approximately 4 kb 3' homologous arm are amplified
by PCR and the fragments are cloned into the targeting vector. Each of
the primers used to amplify the arms is synthesized to contain the
recognition site for a rare-cutting restriction enzyme, positioned at the
5' end of each oligonucleotide. The incorporation of restriction sites
into the arms during PCR amplification facilitates cloning of the
resultant fragments into the standard targeting vector, as the vector
contains mutually compatible unique sites for each enzyme. In the case of
BACH, the primers are designed as listed in the sequence table below. The
cloning enzymes used are as follows: 5'armF XhoI, 5'armR SpeI, 3'armF
AscI and 3'armR FseI. The fragments are cloned in to the vector known as
pTK3IBLMNL depicted in FIG. 3.

[0333]In addition to the arm primer pairs (5'armF/5'armR and
3'armF/3'armR), primers specific to the BACH locus are also designed for
the following purposes: 5' and 3' probe primer pairs (5'prF/5'prR and
3'prF/3'prR) to amplify a short 200-300 bp fragment of non-repetitive
genomic DNA external to and extending beyond each arm, to allow Southern
analysis of the targeted locus, in isolated putative targeted clones; a
mouse genotyping primer pair (+/-F and +/-R) which allows differentiation
between wild-type, heterozygote and homozygous mice; and lastly, a target
screening primer (5'scr) which anneals upstream of the beginning of the
5' arm region, and which produces a target event specific 1.2 kb amplimer
when paired with a primer specific to the 5' end of the vector (DR1).
This amplimer can only be derived from template DNA from cells where the
desired genomic alteration has occurred and allows the identification of
correctly targeted cells from the background of clones containing
randomly integrated copies of the vector.

[0334]The position of the homology arms is chosen to functionally disrupt
the BACH gene by deleting the seven transmembrane spanning regions. A
targeting vector is prepared where the deleted BACH sequence is replaced
with non-homologous sequences composed of an endogenous gene expression
reporter (a frame independent fusion with lacZ) upstream of a selection
cassette composed of promoted neomycin phosphotransferase (neo) gene in
the same orientation as the BACH gene.

[0335]Recombination of the vector into the murine genome at the correct
locus generates an allele for BACH with the structure depicted in FIG. 4.
For comparison, the structure of a wild-type allele for BACH is show in
FIG. 5. The sequence of the entire genomic contig used in this strategy
to prepare the transgenic BACH knock-out mouse is depicted in a separate
section below. Regions corresponding to the primers are annotated.

[0336]It should be noted that the entire 7tm region has been removed in
the knockout allele (FIG. 4). The sites for the enzyme EcoRI are included
as this enzyme is used to confirm the correct integration of the
targeting construct using Southern analysis of putative clones.

[0337]Once the 5' and 3' homology arms are cloned into the targeting
vector, a large highly pure DNA preparation is made using standard
molecular biology techniques. 20 μg of the freshly prepared endotoxin
free DNA is restricted with another rare-cutting restriction enzyme,
PmeI, present at a unique site in the vector backbone between the
amplicillin resistance gene and the bacterial origin of replication. The
linearized DNA is then precipitated and resuspended in 100 μl of
Phosphate Buffered Saline, ready for electroporation.

[0339]24 hours following electroporation the transfected cells are
cultured for 9 days in medium containing 200 μg/ml neomycin. Clones
are picked into 96 well plates, replicated and expanded before being
screened by PCR (using primers 5'scr and DR1, as described above) to
identify clones in which homologous recombination had occurred between
the endogenous BACH gene and the targeting construct. From 200 picked
clones 7 targets are identified. These clones are expanded to allow
replicas to be frozen and sufficient high quality DNA to be prepared for
Southern blot confirmation of the targeting event using the external 5'
and 3' probes prepared as described above, all using standard procedures
(Russ et al, 2000)

Generation of BACH GPCR Deficient Mice

[0340]C57BL/6 female and male mice are mated and blastocysts are isolated
at 3.5 days of gestation. 10-12 cells from a chosen clone are injected
per blastocyst and 7-8 blastocysts are implanted in the uterus of a
pseudopregnant F1 female. Five chimeric pups are born of which one male
is 100% agouti (indicating cells descendent from the targeted clone).
This male chimera is mated with female and MF1 and 129 mice, and germline
transmission is determined by the agouti coat colour and by PCR
genotyping respectively.

[0341]Genotyping is carried out by PCR on lysed tail clips, using the
primers +/-F and +/-R with a third, neo specific primer (neo240F
GTCGTGACCCATGGCGATGCCTGCTTG). This multiplex PCR allows amplification
from the wild-type locus (if present) from primers +/-F and +/-R giving a
243 by band. The site for +/-F is deleted in the knockout mice, so this
amplification will fail. However, the neo240F primer will amplify a 404
bp band from the targeted locus, in combination with the +/-R primer
which anneals to a region just inside the 3' arm. Wild-type samples will
exhibit a single 243 bp band, heterozygous DNA samples yield two bands at
243 bp and 404 bp, and the homozygous samples will show only the target
specific 404 bp band.

LacZ Staining

[0342]The X gal staining of dissected tissues is performed in the
following manner.

[0345]Mice are housed under a 12 h lights-12 h dark light schedule
(lights-on at 6 am) with free access to food and water. Mice (n=12), of
mixed sexes, aged 3 to 4 months old, are submitted to behavioural testing
during the morning, between 10 h and 13 h to avoid any differential
circadian effect on the test results.

Example 2

Expression of Recombinant BACH Protein

[0346]Recombinant BACH is expressed and purified. Two systems are used for
expression.

pTOPO-Echo Donor Based Construct

[0347]A polynucleotide having the sequence shown in SEQ ID NO: 6 is
obtained from the mouse BACH nucleic acid sequence (SEQ ID NO: 5). The
SEQ ID NO: 6 polynucleotide is cloned into a pTOPO-Echo Donor vector
module (Invitrogen pUniV5/His Cat #ET001-10). Transfection of the
resulting construct into a host strain and induction of expression
(according to the manufacturer's instructions) yields a fusion protein
having the sequence of SEQ ID NO: 7.

[0349]A polynucleotide having the sequence shown in SEQ ID NO: 8 is
amplified by PCR using the oligonucleotide primers
AAATATAAGGATCCAGACGATGTTAGCCAACAGCTCC and
TTCGTGAATTCGAGGGCGGAATCCTGGGGACACTG to incorporate new restriction sites,
BamHI and EcoRI at the 5-prime and 3-prime ends of BACH. This is then
digested and ligated into similarly digested pcDNA 3.1A Myc/His
(Invitrogen Cat #V800-20) to incorporate a novel Kozak consensus sequence
(residues 1 to 5 of SEQ ID NO: 8) at the 5' end to improve levels of
expression in mammalian cells.

[0350]The resulting construct is used for high level expression in CHO-K1
cells, and other mammalian cell lines, under the control of the cmv
promoter to yield a fusion polypeptide with C terminal Myc tag (single
underline) and His tag (double underline) to aid detection and
purification. The resultant expressed fusion polypeptide has a sequence
shown in SEQ ID NO: 9.

[0352]Both transient and stable transfection of these cells is achieved.
In transient expression the cells are transfected by lipofection using a
large amount of vector which results in a short-lived fast expression of
the protein. In a stable transfection, the vector, which includes a
selectable marker for neomycin resistance becomes stably integrated into
the genome of the host cell resulting in a long lived cell line with a
high expression level of BACH.

[0353]Cells expressing recombinant BACH are used for assay development,
antibody production, and other purposes as described.

Expression in Other Host Cells

[0354]The recombinant/fusion clone SEQ ID NO: 6 is recombined into a
pBAD-Thio-E fector (Invitrogen Cat #ET100-01) for high level bacterial
expression under control of the araBAD promoter, using a Cre/Lox mediated
recombination system.

[0356]The recombinant/fusion clone SEQ ID NO: 6 is recombined into pcDNA
3.1-E (Invitrogen Cat #ET400-01), using a Cre/Lox mediated recombination
system, for high level expression in CHO-K1 (Chinese Hamster Ovary)
cells, and other mammalian cell lines, under the control of the cmv
promoter.

Example 3

Anti-BACH Antibodies

[0357]Monoclonal and polyclonal antibodies which react with BACH protein
are produced.

Anti-Peptide Antibodies

[0358]The peptide_CRYRDLEVRL, corresponding to amino acids 165-174 of the
full-length BACH polypeptide, is synthesised. The synthetic peptide is
injected into sheep to raise polyclonal anti-peptide antibodies. The
antibodies are purified by standard methods, and are found to bind to
BACH protein in Western blots and ELISA assays.

[0359]Injection into rabbits results in rabbit anti-peptide antibodies,
which are found to bind to BACH protein in Western blots and ELISA
assays.

Whole Cell Preparations as Antigens

[0360]Complete cells expressing a BACH fusion protein, as described above,
are used as a source of BACH epitopes in the immunisation. Injection of
such cells into sheep results in antibodies which are found to bind to
BACH protein in Western blots and ELISA assays. Complete cells are also
injected into mice and rabbits for production of anti-BACH antibodies
from these organisms.

[0361]Complete cells which express the BACH protein in its native form are
also used as antigens for injection of sheep, mice and rabbits for the
production of polyclonal antibodies.

Partially Purified Antigens

[0362]Crude fractions of cells expressing BACH as a native or fusion
protein are made. Membrane fractions are found to include BACH protein
and are used to inject sheep for production of anti-BACH antibodies.
These antibodies are found to react to BACH protein in Western blots and
ELISA assays. Similarly, injection of mice and rabbits results in
anti-BACH antibodies which react to BACH protein.

Purified Antigens

[0363]The BACH protein is produced in its native form or as a fusion by
the methods described above. BACH protein is purified using affinity
chromatography, using the appropriate purification procedure. For
example, nickel agarose resins are used to purify His-tagged fusion
proteins. If necessary, the fusion protein is cleaved using specific
proteases to yield the native protein. Both the tagged polypeptide and
the purified cleaved product are used to raise antibodies in a variety of
species including sheep, rabbits, mice and goats. These antibodies are
found to react to BACH protein.

Example 4

Expression of BACH in the Dorsal Root Ganglion and the Trigeminal Ganglion

[0364]BACH expression is detected in the dorsal root ganglion and the
trigeminal ganglion, as shown in FIGS. 6 and 7.

[0365]The trigeminal nerve is the sensory supply to the face, the greater
part of the scalp, the teeth, the oral and nasal cavities, the dura
matter and the cerebral blood vessels.

[0366]It gives the motor supply to the masticatory muscles, and contains
proprioceptive fibers from the masticatory and probably the extraocular
and facial muscles. The opthalmic nerve, where most of the staining was
observed, is wholly sensory. It supplies the eyeball, lacrimal gland and
conjunctiva, part of the nasal mucosa and the skin of the nose, eyelids,
forehead and part of the scalp. Fibres joining the trigeminal from the
facial are afferent and arise largely from facial musculature, a minority
being proprioceptive, the majority pain.

[0367]Lac Z expression is observed on the dorsal surface of the ganglion
itself (therefore not on the motor division of the ganglion), with an
extension towards the opthalmic and mandibular/maxillary divisions. No
staining is observed in the opthalmic, mandibular and maxillary nerves
themselves.

[0368]Staining in the tongue, the nasal region, the conjuctiva, harderian
gland, all are consistent with the trigeminal ganglion's inervation
processes. They also favour a role for BACH in pain/sensitivity of the
face. Therefore, BACH mutants have modified sensitivity of the face,
sense of smell, of taste, or audition.

[0369]Examination of the expression pattern and analgesia phenotypic data
of the mutant mice show that BACH is a target for potential treatment of
trigeminal neuralgia as well as some type of migraine. Orofacial pain is
a consequence of trigeminal neuralgia, in which paroxysmal pain radiates
over one, or two divisions of the trigeminal nerve. The opthalmic
division is rarely affected. Drug treatment is usually effective but if
it fails surgical treatment is used. None of these surgical treatments
has proved satisfactory. No specific drug has been developed yet.

Example 5

Expression of BACH in the Vestibulocochlear Nerve

[0370]Staining is also observed in cranial nerve number 8
(vestibulocochlear) (as it comes out from the skull). P2X receptors are
expression in this nerve (Xiang 19991).

[0371]This nerve contains 2 major sets of afferent fibers transmitting
impulses from the inner ear to the brain. Lesions to this nerve may cause
imbalance. We actually observe several mutants inbred mice having
difficulty during the wire balance maneuver test as well as walking
backwards, which is an abnormal hallucinatory behaviour (mice walk
backward result from an apparent hallucination of sliding down an
inclined plane), and this was much worse when the mice where submitted to
the visual cliff test, probably because the chequered pattern accentuated
the hallucination.

Example 6

Expression of BACH in the Cerebellum

[0372]LacZ staining in cerebellum of the -/- mice was detected in a
distinct layer of the cerebellum. The cerebellum links some of the major
sensory and motor areas of the CNS. Indeed its main function is for motor
learning and reflex modification. The output of the cerebellum is almost
entirely to areas of the brain that control movement. Consequently, the
rotarod tests that show the -/- mice have improved motor learning and
function would almost certainly have altered signaling in the cerebellum.
This is supported by the observation of discreet staining in the
cerebellum. Application of PPADS, the P2 antagonists to mice resulted in
aggressiveness, hyporeactivity and immobility indicating that these
receptors may be involved in age related impaired movement disorders.
Although it is believed that P2X receptors are also involved.

Example 7

Analysis of Blood Samples from BACH Knock-Out Mice

[0373]Analysis of blood samples taken from mutant BACH animals shows an
increased level of circulating cholesterol. This coupled with the
expression seen in the liver, gall bladder and in fat around the heart
may imply a use for the BACH protein in the development of therapeutics
to treat hypercholesterolaemia, dislipdaemias, obesity or drugs to affect
energy regulation.

[0374]Finally, by comparing with the role and localisation of purine
receptors (e.g. P2Y12 regulate mucine expression on the conjuctiva), BACH
receptors may also be employed as potential targets for developing drug
relating to dry-eye disorders, cystic fibrosis, hyperactive bladder,
hypercholesterolaemia, dislipdaemias and obesity.

Example 8

Motor Control and Balance in BACH Knock-Out Mouse

[0375]BACH protein has a role in motor co-ordination and balance that can
be demonstrated by their performance on the Rotarod apparatus.

[0376]Motor co-ordination and balance are measured by performance on the
rotarod. We use an accelerating Rotarod (Ugo Basile, Linton instruments,
Jones and Roberts 19681). We also use the Rotarod to assess motor
learning by repeating the task over several days.

[0377]Mice are placed on the Rotarod, which accelerates at a constant rate
from 4 to 40 rpm in 5 in. As the Rotarod reaches higher speed, the mice
often grip the Rotarod and hang on for a full rotation (i.e. passive
rotation). The time at which the mouse makes one full rotation is
recorded. Mice are left on the Rotarod after the first passive rotation
and allowed to perform the rest of the test until they drop from the rod.
Mice are given 3 trials on three consecutive days.

[0378]Rotarod testing reveals better performance in mutants than wild type
animals in both the outbred and inbred background (p=0.08). The outbred
mutants also shown better motor learning abilities (FIG. 8A)
(P<0.001). However, this was not observed in inbred animals (FIG. 8B).

Example 9

Visual Ability, Anxiety and Mobility in BACH Knock-Out Mouse

[0379]Tests for visual ability, anxiety and mobility in a BACH knock-out
mouse are conducted using a visual cliff test.

[0380]The Visual cliff was developed by Fox (19651) and provides a
measure of gross visual ability. It evaluates the ability of the mouse to
see the drop-off at the edge of a horizontal surface. Time for the animal
to move one placed on the cliff (latency) is a measure of anxiety.

[0381]A Perspex box is built with a horizontal plane connected to a
vertical drop of 0.5 m. A black and white chequerboard pattern
accentuates the vertical drop-out. A sheet of clear Perspex is placed
across the top horizontal cliff, extending over the top, thus there is
the visual appearance of a cliff, but in fact the Perspex provides a
solid horizontal surface.

[0382]Each mouse is given 10 trials on the visual cliff. A trial consists
of placing the mouse on the centre `ridge` and noting the time taken from
the animal to move off the `ridge` (latency) and recording the side on
which the mouse stepped. The result is considered positive when the
animal chooses to walk on the chequered "safe" side and avoids the cliff
and negative result for the other way round. After 5 trials the box is
turned through 180 degrees and a further 5 trials given. This is done to
eliminate the variable of the position of the observer. The platform is
cleaned between each animal.

[0383]Latency ranges from 1 second to 7 min and there is a trend towards a
longer latency in mutants (P=0.059). Wild type and mutant animals perform
relatively well on the task, with consistent positive results. In
heterozygous animals however, results are only positive in 55% of the
trials, which is consistent with the kind of results observed with blind
mice (Fox, 19651) (Table 2). However, there are at least two
alternative explanations for this: Firstly, to avoid impairing other
tasks, we did not remove the vibrissaes in our mice, and mice are known
to use these to navigate. Secondly, we observe that some mice seem
somewhat "nervous" during the visual cliff test, displaying either
freezing behaviour or alternatively trying to escape the hand of the
observer regardless of the cliff whilst being lowered onto the ridge.
Although we did not find any significant difference between genotypes in
the locomotor behaviour recorded in the arena or the struggle during
handling during the Shirpa screen, this could have affected the
responses.

[0384]As can be seen from the above Tables 2 and 3, BACH mutants have a
longer latency period in the visual cliff test when compared to wild type
mice, which indicates among other conditions a movement related disorder.

[0385]BACH mutants also demonstrate retropulsion (walking backwards). This
is accentuated when placed in the visual cliff arena believed to be due
to the hallucination of falling forward. This indicates that loss of
functional BACH results in movement disorders and dyskinesias.

[0386]BACH mutants are seen to be less sensitive to touch and pain stimuli
using a range of tests. The tests in this and the following four Examples
assess both neuropathic and inflammatory pain.

[0387]Skin sensitivity is assessed by applying pressure on the hindpaw
with a sharpened dowel rod whilst the animal is resting on a grid.
Responses are graded as follow: 0=no withdrawal; 1=slow withdrawal of the
paw; 2=medium withdrawal of the paw; 3=fast withdrawal of the paw.

[0388]FIG. 9 shows results of the paw pressure test. Using the paw
pressure test BACH mutants are seen to be less sensitive to pressure and
pain than their wild-type counterparts.

[0389]A tail flick analgesia test is performed using a Tail-Flick
Analgesia Meter. This equipment provides an easy to use method to
determine pain sensitivity accurately and reproducibly in rodents
(D'Amour and Smith, 1941). The instrument has a shutter-controlled lamp
as a heat source. The lamp is located below the animal to provide a less
confining environment. Tail flick is detected by the automatic detection
circuitry, which leaves the user's hands free to handle the animal. The
animal is restrained in a ventilated tube and its tail placed on a
sensing groove on top of the equipment.

[0390]Activation of an intense light beam to the tail through opening of
the shutter results in discomfort at some point when the animal will
flick its tail out of the beam. In the automatic mode a photo-detector
detects the tail motion causing the clock to stop and the shutter to
close. The total time elapsed between the shutter opening and the
animal's reaction is recorded.

[0391]Responses of mutant transgenic mice are compared with age and sex
matched wild-type mice. A single animal may be subjected to different
heat settings to produce an increase in tail temperature no greater than
55° C.

[0392]Using the tail flick test BACH mutants are shown to be less
sensitive to heat induced pain than their wild-type counterparts and
withdraw their tails after a longer time period of exposure to the heat
source.

[0393]The formalin test measures the response to a noxious substances
injected into a hind paw. A volume of 20 μl of a 1% formalin solution
is injected through a fine gauge needle subcutaneously into the dorsal
surface of one hindpaw. Licking and biting the hindpaw is quantitated as
cumulative number of seconds engaged in the behaviours. A rating scale is
used: 1=the formalin injected paw rests lightly on the floor bearing less
weight; 2=the injected paw is elevated; 3=the injected paw is licked,
bitten or shaken.

[0394]Two phases of responses are seen in the formalin test. Phase 1
begins immediately after injection and lasts about 10 mins, representing
the acute burst of activity from pain fibres. Phase two begins about 20
mins after injection and continues for about one hour. This phase appears
to represent responses to tissue damage, including inflammatory
hyperalgesia.

[0395]Using the formalin test BACH mutants are shown to be less sensitive
to inflammatory pain and show a reduced severity of response in the
formalin test when compared to wild type animals.

[0396]A test for touch, which is used to measure pain thresholds, employs
von Frey hairs. These hairs are a set of very fine gauge calibrated
wires. Withdrawal threshold to mechanical stimulation is measured. The
animal stands on an elevated platform in which the surface is a wide
gauge wire mesh. The Von Frey hair is inserted from below, up through the
holes in the mesh, to poke the undersurface of the hindpaw. At threshold,
the mouse responds by flicking its paw away from the hair, generally
followed by raising the paw, licking the paw, and or vocalisation.
Mechanical withdrawal threshold is defined as the minimum gauge wire
stimulus that elicits withdrawal reactions in two out of three
consecutive trials.

[0397]In the Von Frey hair test for touch, mutant BACH mice are seen to
have a higher mechanical withdrawal threshold demonstrating that they are
less sensitive to the stimuli.

[0398]BACH is expressed in the bladder and by measuring the frequency of
micturation and the volume of fluid released. Loss of this protein is
shown to result in abnormal bladder motility. BACH mutants have
hypoactive bladder urinating less often but releasing a larger volume and
suffer urinary retention. It is likely that BACH plays a role in erectile
dysfunction and the control of motor fibres in the prostate.

Example 15

Identification of BACH Regulatory Elements

[0399]During evolution genes undergo sequence divergence. Sequences that
affect the function of a gene tend to be better conserved and these
include not only coding sequences but also promoter control element and
enhancers. By identification of homologues and orthologues of BACH in
multiple species and comparison of the promoter regions such control
elements and enhancers are located.

[0400]To find human BACH control elements and enhancers we compare the
promoter regions from human BACH to the promoter region of BACH from the
pufferfish (Takifugu rubripes) using Blast type alignments. Conserved
patches of sequence are the critical elements that control the level, the
timing and the location of BACH gene expression.

[0401]Human populations and disease cohorts are then screened for
polymorphisms and in the control regions identified in this manner.
Mutations, such as SNPs, in these regions will affect the control of BACH
gene expression and will cause conditions such as BACH associated
diseases. Similar mutations are generated in the promoter sequences and
used to assess their effect on either BACH or a reporter gene expression
in cell lines or transgenic mice. Polymorphisms that are shown to have a
functional effect are then used in diagnostic screens for BACH specific
diseases conditions outlined.

[0425]Each of the applications and patents mentioned in this document, and
each document cited or referenced in each of the above applications and
patents, including during the prosecution of each of the applications and
patents ("application cited documents") and any manufacturer's
instructions or catalogues for any products cited or mentioned in each of
the applications and patents and in any of the application cited
documents, are hereby incorporated herein by reference. Furthermore, all
documents cited in this text, and all documents cited or referenced in
documents cited in this text, and any manufacturer's instructions or
catalogues for any products cited or mentioned in this text, are hereby
incorporated herein by reference.

[0426]Various modifications and variations of the described methods and
system of the invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention. Although
the invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed should
not be unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention which
are obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the claims.

[0427]Mouse BACH Genomic Sequence (SEQ ID NO: 10)

[0428]The genomic sequence of mouse BACH is shown below (SEQ ID NO: 10);
this corresponds to the entire genomic contig used to prepare BACH
knockout mice. Regions corresponding to the primers are annotated.

[0429]Where the term "a mouse BACH genomic sequence" is used in this
document, it should be understood as a reference to this sequence.